Multiplex PCR Tests for Detection of Pathogens Associated with Gastroenteritis

PubMed Central

Zhang, Hongwei; Morrison, Scott; Tang, Yi-Wei

2016-01-01

Synopsis A wide range of enteric pathogens can cause infectious gastroenteritis. Conventional diagnostic algorithms including culture, biochemical identification, immunoassay and microscopic examination are time consuming and often lack sensitivity and specificity. Advances in molecular technology have as allowed its use as clinical diagnostic tools. Multiplex PCR based testing has made its way to gastroenterology diagnostic arena in recent years. In this article we present a review of recent laboratory developed multiplex PCR tests and current commercial multiplex gastrointestinal pathogen tests. We will focus on two FDA cleared commercial syndromic multiplex tests: Luminex xTAG GPP and Biofire FimArray GI test. These multiplex tests can detect and identify multiple enteric pathogens in one test and provide results within hours. Multiplex PCR tests have shown superior sensitivity to conventional methods for detection of most pathogens. The high negative predictive value of these multiplex tests has led to the suggestion that they be used as screening tools especially in outbreaks. Although the clinical utility and benefit of multiplex PCR test are to be further investigated, implementing these multiplex PCR tests in gastroenterology diagnostic algorithm has the potential to improve diagnosis of infectious gastroenteritis. PMID:26004652

Establishment of a nested-ASP-PCR method to determine the clarithromycin resistance of Helicobacter pylori.

PubMed

Luo, Xiao-Feng; Jiao, Jian-Hua; Zhang, Wen-Yue; Pu, Han-Ming; Qu, Bao-Jin; Yang, Bing-Ya; Hou, Min; Ji, Min-Jun

2016-07-07

To investigate clarithromycin resistance positions 2142, 2143 and 2144 of the 23SrRNA gene in Helicobacter pylori (H. pylori) by nested-allele specific primer-polymerase chain reaction (nested-ASP-PCR). The gastric tissue and saliva samples from 99 patients with positive results of the rapid urease test (RUT) were collected. The nested-ASP-PCR method was carried out with the external primers and inner allele-specific primers corresponding to the reference strain and clinical strains. Thirty gastric tissue and saliva samples were tested to determine the sensitivity of nested-ASP-PCR and ASP-PCR methods. Then, clarithromycin resistance was detected for 99 clinical samples by using different methods, including nested-ASP-PCR, bacterial culture and disk diffusion. The nested-ASP-PCR method was successfully established to test the resistance mutation points 2142, 2143 and 2144 of the 23SrRNA gene of H. pylori. Among 30 samples of gastric tissue and saliva, the H. pylori detection rate of nested-ASP-PCR was 90% and 83.33%, while the detection rate of ASP-PCR was just 63% and 56.67%. Especially in the saliva samples, nested-ASP-PCR showed much higher sensitivity in H. pylori detection and resistance mutation rates than ASP-PCR. In the 99 RUT-positive gastric tissue and saliva samples, the H. pylori-positive detection rate by nested-ASP-PCR was 87 (87.88%) and 67 (67.68%), in which there were 30 wild-type and 57 mutated strains in gastric tissue and 22 wild-type and 45 mutated strains in saliva. Genotype analysis showed that three-points mixed mutations were quite common, but different resistant strains were present in gastric mucosa and saliva. Compared to the high sensitivity shown by nested-ASP-PCR, the positive detection of bacterial culture with gastric tissue samples was 50 cases, in which only 26 drug-resistant strains were found through analyzing minimum inhibitory zone of clarithromycin. The nested-ASP-PCR assay showed higher detection sensitivity than ASP-PCR and drug sensitivity testing, which could be performed to evaluate clarithromycin resistance of H. pylori.

Establishment of a nested-ASP-PCR method to determine the clarithromycin resistance of Helicobacter pylori

PubMed Central

Luo, Xiao-Feng; Jiao, Jian-Hua; Zhang, Wen-Yue; Pu, Han-Ming; Qu, Bao-Jin; Yang, Bing-Ya; Hou, Min; Ji, Min-Jun

2016-01-01

AIM: To investigate clarithromycin resistance positions 2142, 2143 and 2144 of the 23SrRNA gene in Helicobacter pylori (H. pylori) by nested-allele specific primer-polymerase chain reaction (nested-ASP-PCR). METHODS: The gastric tissue and saliva samples from 99 patients with positive results of the rapid urease test (RUT) were collected. The nested-ASP-PCR method was carried out with the external primers and inner allele-specific primers corresponding to the reference strain and clinical strains. Thirty gastric tissue and saliva samples were tested to determine the sensitivity of nested-ASP-PCR and ASP-PCR methods. Then, clarithromycin resistance was detected for 99 clinical samples by using different methods, including nested-ASP-PCR, bacterial culture and disk diffusion. RESULTS: The nested-ASP-PCR method was successfully established to test the resistance mutation points 2142, 2143 and 2144 of the 23SrRNA gene of H. pylori. Among 30 samples of gastric tissue and saliva, the H. pylori detection rate of nested-ASP-PCR was 90% and 83.33%, while the detection rate of ASP-PCR was just 63% and 56.67%. Especially in the saliva samples, nested-ASP-PCR showed much higher sensitivity in H. pylori detection and resistance mutation rates than ASP-PCR. In the 99 RUT-positive gastric tissue and saliva samples, the H. pylori-positive detection rate by nested-ASP-PCR was 87 (87.88%) and 67 (67.68%), in which there were 30 wild-type and 57 mutated strains in gastric tissue and 22 wild-type and 45 mutated strains in saliva. Genotype analysis showed that three-points mixed mutations were quite common, but different resistant strains were present in gastric mucosa and saliva. Compared to the high sensitivity shown by nested-ASP-PCR, the positive detection of bacterial culture with gastric tissue samples was 50 cases, in which only 26 drug-resistant strains were found through analyzing minimum inhibitory zone of clarithromycin. CONCLUSION: The nested-ASP-PCR assay showed higher detection sensitivity than ASP-PCR and drug sensitivity testing, which could be performed to evaluate clarithromycin resistance of H. pylori. PMID:27433095

Rapid and sensitive insulated isothermal PCR for point-of-need feline leukaemia virus detection.

PubMed

Wilkes, Rebecca P; Anis, Eman; Dunbar, Dawn; Lee, Pei-Yu A; Tsai, Yun-Long; Lee, Fu-Chun; Chang, Hsiao-Fen G; Wang, Hwa-Tang T; Graham, Elizabeth M

2018-04-01

Objectives Feline leukaemia virus (FeLV), a gamma retrovirus, causes diseases of the feline haematopoietic system that are invariably fatal. Rapid and accurate testing at the point-of-need (PON) supports prevention of virus spread and management of clinical disease. This study evaluated the performance of an insulated isothermal PCR (iiPCR) that detects proviral DNA, and a reverse transcription (RT)-iiPCR that detects both viral RNA and proviral DNA, for FeLV detection at the PON. Methods Mycoplasma haemofelis, feline coronavirus, feline herpesvirus, feline calicivirus and feline immunodeficiency virus were used to test analytical specificity. In vitro transcribed RNA, artificial plasmid, FeLV strain American Type Culture Collection VR-719 and a clinical FeLV isolate were used in the analytical sensitivity assays. A retrospective study including 116 clinical plasma and serum samples that had been tested with virus isolation, real-time PCR and ELISA, and a prospective study including 150 clinical plasma and serum samples were implemented to evaluate the clinical performances of the iiPCR-based methods for FeLV detection. Results Ninety-five percent assay limit of detection was calculated to be 16 RNA and five DNA copies for the RT-iiPCR, and six DNA copies for the iiPCR. Both reactions had analytical sensitivity comparable to a reference real-time PCR (qPCR) and did not detect five non-target feline pathogens. The clinical performance of the RT-iiPCR and iiPCR had 98.82% agreement (kappa[κ] = 0.97) and 100% agreement (κ = 1.0), respectively, with the qPCR (n = 85). The agreement between an automatic nucleic extraction/RT-iiPCR system and virus isolation to detect FeLV in plasma or serum was 95.69% (κ = 0.95) and 98.67% (κ = 0.85) in a retrospective (n = 116) and a prospective (n = 150) study, respectively. Conclusions and relevance These results suggested that both RT-iiPCR and iiPCR assays can serve as reliable tools for PON FeLV detection.

PCR-based study of conserved and variable DNA sequences of Tritrichomonas foetus isolates from Saskatchewan, Canada.

PubMed Central

Riley, D E; Wagner, B; Polley, L; Krieger, J N

1995-01-01

The protozoan parasite Tritrichomonas foetus causes infertility and spontaneous abortion in cattle. In Saskatchewan, Canada, the culture prevalence of trichomonads was 65 of 1,048 (6%) among 1,048 bulls tested within a 1-year period ending in April 1994. Saskatchewan was previously thought to be free of the parasite. To confirm the culture results, possible T. foetus DNA presence was determined by the PCR. All of the 16 culture-positive isolates tested were PCR positive by a single-band test, but one PCR product was weak. DNA fingerprinting by both T17 PCR and randomly amplified polymorphic DNA PCR revealed genetic variation or polymorphism among the T. foetus isolates. T17 PCR also revealed conserved loci that distinguished these T. foetus isolates from Trichomonas vaginalis, from a variety of other protozoa, and from prokaryotes. TCO-1 PCR, a PCR test designed to sample DNA sequence homologous to the 5' flank of a highly conserved cell division control gene, detected genetic polymorphism at low stringency and a conserved, single locus at higher stringency. These findings suggested that T. foetus isolates exhibit both conserved genetic loci and polymorphic loci detectable by independent PCR methods. Both conserved and polymorphic genetic loci may prove useful for improved clinical diagnosis of T. foetus. The polymorphic loci detected by PCR suggested either a long history of infection or multiple lines of T. foetus infection in Saskatchewan. Polymorphic loci detected by PCR may provide data for epidemiologic studies of T. foetus. PMID:7615746

Detection of adenoviruses in shellfish by means of conventional-PCR, nested-PCR, and integrated cell culture PCR (ICC/PCR).

PubMed

Rigotto, C; Sincero, T C M; Simões, C M O; Barardi, C R M

2005-01-01

We tested three PCR based methodologies to detect adenoviruses associated with cultivated oysters. Conventional-PCR, nested-PCR, and integrated cell culture-PCR (ICC/PCR) were first optimized using oysters seeded with know amounts of Adenovirus serotype 5 (Ad5). The maximum sensitivity for Ad5 detection was determined for each method, and then used to detect natural adenovirus contamination in oysters from three aquiculture farms in Florianopolis, Santa Catarina State, Brazil, over a period of 6 months. The results showed that the nested-PCR was more sensitive (limit of detection: 1.2 PFU/g of tissue) than conventional-PCR and ICC-PCR (limit of detection for both: 1.2 x 10(2)PFU/g of tissue) for detection of Ad5 in oyster extracts. Nested-PCR was able to detect 90% of Ad5 contamination in harvested oyster samples, while conventional-PCR was unable to detect Ad5 in any of the samples. The present work suggests that detection of human adenoviruses can be used as a tool to monitor the presence of human viruses in marine environments where shellfish grow, and that nested-PCR is the method of choice.

[The establishment of a novel method of nano-immunomagnetic separation and Real-time PCR for detecting Vibrio cholerae from seafood].

PubMed

Cheng, Jinxia; Zeng, Jing; Liu, Li; Wei, Haiyan; Zhao, Xiaojuan; Zhang, Ximeng; Zhang, Lei; Zhang, Haiyu

2014-02-01

A novel method of Nano-Immunomagnetic Separation (Nano-IMS) plus Real-time PCR was established for detecting Vibrio cholerae. The Nano-Immunomagnetic Beads were created by using the monoclonal antibody of Vibrio cholerae, which was named Nano-IMB-Vc. Nano-IMB-Vc has specific adsorption of Vibrio cholerae, combined with Real-time PCR technology, a method for rapid detection of Vibrio cholerae was established. The capture specificity of Nano-IMB-Vc was tested by using 15 bacteria strains. The specificity of Real-time PCR method was tested by using 102 targets and 101 non-targets bacteria strains. The sensitivity of Nano-IMS plus Real-time PCR were tested in pure culture and in artificial samples and compared with NMKL No.156. The capture ratio of Nano-IMB-Vc was reached 70.2% at the level of 10(3) CFU/ml. In pure culture, the sensitivity of Nano-IMS plus Real-time PCR was reached at 5.4×10(2) CFU/ml. The specific of Real-time PCR method was tested by using 102 targets and 101 non-targets bacteria. The results showed that 102 strains of Vibrio cholerae test results were all positive, and the rest of the 101 strains of non-target bacteria test results were negative. No cross-reaction was founded. Add 1 CFU vibrio cholerae per 25 g sample, it could be detect with Nano-IMS plus Real-time PCR method after 8 hours enrichment. The Nano-IMS plus Real-time PCR method of Vibrio cholerae established in this study has good specificity and sensitivity, which could be applied to the rapid detection of Vibrio cholerae.

Direct detection of Mycobacterium tuberculosis complex in bovine and bubaline tissues through nested-PCR.

PubMed

Araújo, Cristina P; Osório, Ana Luiza A R; Jorge, Klaudia S G; Ramos, Carlos A N; Souza Filho, Antonio F; Vidal, Carlos E S; Vargas, Agueda P C; Roxo, Eliana; Rocha, Adalgiza S; Suffys, Philip N; Fonseca, Antônio A; Silva, Marcio R; Barbosa Neto, José D; Cerqueira, Valíria D; Araújo, Flábio R

2014-01-01

Post-mortem bacterial culture and specific biochemical tests are currently performed to characterize the etiologic agent of bovine tuberculosis. Cultures take up to 90 days to develop. A diagnosis by molecular tests such as PCR can provide fast and reliable results while significantly decreasing the time of confirmation. In the present study, a nested-PCR system, targeting rv2807, with conventional PCR followed by real-time PCR, was developed to detect Mycobacterium tuberculosis complex (MTC) organisms directly from bovine and bubaline tissue homogenates. The sensitivity and specificity of the reactions were assessed with DNA samples extracted from tuberculous and non-tuberculous mycobacteria, as well as other Actinomycetales species and DNA samples extracted directly from bovine and bubaline tissue homogenates. Regarding the analytical sensitivity, DNA of the M. bovis AN5 strain was detected up to 1.5 pg by nested-PCR, whereas DNA of M. tuberculosis H37Rv strain was detected up to 6.1 pg. The nested-PCR system showed 100% analytical specificity for MTC when tested with DNA of reference strains of non-tuberculous mycobacteria and closely-related Actinomycetales. A clinical sensitivity level of 76.7% was detected with tissues samples positive for MTC by means of the culture and conventional PCR. A clinical specificity of 100% was detected with DNA from tissue samples of cattle with negative results in the comparative intradermal tuberculin test. These cattle exhibited no visible lesions and were negative in the culture for MTC. The use of the nested-PCR assay to detect M. tuberculosis complex in tissue homogenates provided a rapid diagnosis of bovine and bubaline tuberculosis.

Direct detection of Mycobacterium tuberculosis complex in bovine and bubaline tissues through nested-PCR

PubMed Central

Araújo, Cristina P.; Osório, Ana Luiza A.R.; Jorge, Klaudia S.G.; Ramos, Carlos A.N.; Souza Filho, Antonio F.; Vidal, Carlos E.S.; Vargas, Agueda P.C.; Roxo, Eliana; Rocha, Adalgiza S.; Suffys, Philip N.; Fonseca, Antônio A.; Silva, Marcio R.; Barbosa Neto, José D.; Cerqueira, Valíria D.; Araújo, Flábio R.

2014-01-01

Post-mortem bacterial culture and specific biochemical tests are currently performed to characterize the etiologic agent of bovine tuberculosis. Cultures take up to 90 days to develop. A diagnosis by molecular tests such as PCR can provide fast and reliable results while significantly decreasing the time of confirmation. In the present study, a nested-PCR system, targeting rv2807, with conventional PCR followed by real-time PCR, was developed to detect Mycobacterium tuberculosis complex (MTC) organisms directly from bovine and bubaline tissue homogenates. The sensitivity and specificity of the reactions were assessed with DNA samples extracted from tuberculous and non-tuberculous mycobacteria, as well as other Actinomycetales species and DNA samples extracted directly from bovine and bubaline tissue homogenates. Regarding the analytical sensitivity, DNA of the M. bovis AN5 strain was detected up to 1.5 pg by nested-PCR, whereas DNA of M. tuberculosis H37Rv strain was detected up to 6.1 pg. The nested-PCR system showed 100% analytical specificity for MTC when tested with DNA of reference strains of non-tuberculous mycobacteria and closely-related Actinomycetales. A clinical sensitivity level of 76.7% was detected with tissues samples positive for MTC by means of the culture and conventional PCR. A clinical specificity of 100% was detected with DNA from tissue samples of cattle with negative results in the comparative intradermal tuberculin test. These cattle exhibited no visible lesions and were negative in the culture for MTC. The use of the nested-PCR assay to detect M. tuberculosis complex in tissue homogenates provided a rapid diagnosis of bovine and bubaline tuberculosis. PMID:25242951

Development of an Internal Positive Control for Rapid Diagnosis of Avian Influenza Virus Infections by Real-Time Reverse Transcription-PCR with Lyophilized Reagents

PubMed Central

Das, Amaresh; Spackman, Erica; Senne, Dennis; Pedersen, Jan; Suarez, David L.

2006-01-01

We developed an internal positive control (IPC) RNA to help ensure the accuracy of the detection of avian influenza virus (AIV) RNA by reverse transcription (RT)-PCR and real-time RT-PCR (RRT-PCR). The IPC was designed to have the same binding sites for the forward and reverse primers of the AIV matrix gene as the target amplicon, but it had a unique internal sequence used for the probe site. The amplification of the viral RNA and the IPC by RRT-PCR were monitored with two different fluorescent probes in a multiplex format, one specific for the AIV matrix gene and the other for the IPC. The RRT-PCR test was further simplified with the use of lyophilized bead reagents for the detection of AIV RNA. The RRT-PCR with the bead reagents was more sensitive than the conventional wet reagents for the detection of AIV RNA. The IPC-based RRT-PCR detected inhibitors in blood, kidney, lungs, spleen, intestine, and cloacal swabs, but not allantoic fluid, serum, or tracheal swabs The accuracy of RRT-PCR test results with the lyophilized beads was tested on cloacal and tracheal swabs from experimental birds inoculated with AIV and compared with virus isolation (VI) on embryonating chicken eggs. There was 97 to 100% agreement of the RRT-PCR test results with VI for tracheal swabs and 81% agreement with VI for cloacal swabs, indicating a high level of accuracy of the RRT-PCR assay. The same IPC in the form of armored RNA was also used to monitor the extraction of viral RNA and subsequent detection by RRT-PCR. PMID:16954228

Evaluation of a Commercial Multiplex PCR for Rapid Detection of Multi Drug Resistant Gram Negative Infections

PubMed Central

Chavada, Ruchir; Maley, Michael

2015-01-01

Introduction: Community and healthcare associated infections caused by multi-drug resistant gram negative organisms (MDR GN) represent a worldwide threat. Nucleic Acid Detection tests are becoming more common for their detection; however they can be expensive requiring specialised equipment and local expertise. This study was done to evaluate the utility of a commercial multiplex tandem (MT) PCR for detection of MDR GN. Methods: The study was done on stored laboratory MDR GN isolates from sterile and non-sterile specimens (n=126, out of stored 567 organisms). Laboratory validation of the MT PCR was done to evaluate sensitivity, specificity and agreement with the current phenotypic methods used in the laboratory. Amplicon sequencing was also done on selected isolates for assessing performance characteristics. Workflow and cost implications of the MT PCR were evaluated. Results: The sensitivity and specificity of the MT PCR were calculated to be 95% and 96.7% respectively. Agreement with the phenotypic methods was 80%. Major lack of agreement was seen in detection of AmpC beta lactamase in enterobacteriaceae and carbapenemase in non-fermenters. Agreement of the MT PCR with another multiplex PCR was found to be 87%. Amplicon sequencing confirmed the genotype detected by MT PCR in 94.2 % of cases tested. Time to result was faster for the MT PCR but cost per test was higher. Conclusion: This study shows that with carefully chosen targets for detection of resistance genes in MDR GN, rapid and efficient identification is possible. MT PCR was sensitive and specific and likely more accurate than phenotypic methods. PMID:26464612

Bayesian estimation of true prevalence, sensitivity and specificity of three diagnostic tests for detection of Escherichia coli O157 in cattle feces.

PubMed

Ekong, Pius S; Sanderson, Michael W; Bello, Nora M; Noll, Lance W; Cernicchiaro, Natalia; Renter, David G; Bai, Jianfa; Nagaraja, T G

2017-12-01

Cattle are a reservoir for Escherichia coli O157 and they shed the pathogen in their feces. Fecal contaminants on the hides can be transferred onto carcasses during processing at slaughter plants, thereby serving as a source of foodborne infection in humans. The detection of E. coli O157 in cattle feces is based on culture, immunological, and molecular methods We evaluated the diagnostic sensitivity and specificity of one culture- and two PCR-based tests for the detection of E. coli O157 in cattle feces, and its true prevalence using a Bayesian implementation of latent class models. A total of 576 fecal samples were collected from the floor of pens of finishing feedlot cattle in the central United States during summer 2013. Samples were enriched and subjected to detection of E. coli O157 by culture (immunomagnetic separation, plating on a selective medium, latex agglutination, and indole testing), conventional PCR (cPCR), and multiplex quantitative PCR (mqPCR). The statistical models assumed conditional dependence of the PCR tests and high specificity for culture (mode=99%; 5th percentile=97%). Prior estimates of test parameters were elicited from three experts. Estimated posterior sensitivity (posterior median and 95% highest posterior density intervals) of culture, cPCR, and mqPCR was 49.1% (44.8-53.4%), 59.7% (55.3-63.9%), and 97.3% (95.1-99.0%), respectively. Estimated posterior specificity of culture, cPCR, and mqPCR were 98.7% (96.8-99.8%), 94.1% (87.4-99.1%), and 94.8% (84.1-99.9%), respectively. True prevalence was estimated at 91.3% (88.1-94.2%). There was evidence of a weak conditional dependence between cPCR and mqPCR amongst test positive samples, but no evidence of conditional dependence amongst test negative samples. Sensitivity analyses showed that overall our posterior inference was rather robust to the choice of priors, except for inference on specificity of mqPCR, which was estimated with considerable uncertainty. Our study evaluates performance of three diagnostic tests for detection of E. coli O157 in feces of feedlot cattle which is important for quantifying true fecal prevalence and adjusting for test error in risk modeling. Copyright © 2017 Elsevier B.V. All rights reserved.

Evaluation of sensitivity of TaqMan RT-PCR for rubella virus detection in clinical specimens.

PubMed

Okamoto, Kiyoko; Mori, Yoshio; Komagome, Rika; Nagano, Hideki; Miyoshi, Masahiro; Okano, Motohiko; Aoki, Yoko; Ogura, Atsushi; Hotta, Chiemi; Ogawa, Tomoko; Saikusa, Miwako; Kodama, Hiroe; Yasui, Yoshihiro; Minagawa, Hiroko; Kurata, Takako; Kanbayashi, Daiki; Kase, Tetsuo; Murata, Sachiko; Shirabe, Komei; Hamasaki, Mitsuhiro; Kato, Takashi; Otsuki, Noriyuki; Sakata, Masafumi; Komase, Katsuhiro; Takeda, Makoto

2016-07-01

An easy and reliable assay for detection of the rubella virus is required to strengthen rubella surveillance. Although a TaqMan RT-PCR assay for detection of the rubella virus has been established in Japan, its utility for diagnostic purposes has not been tested. To allow introduction of the TaqMan RT-PCR into the rubella surveillance system in Japan, the sensitivity of the assay was determined using representative strains for all genotypes and clinical specimens. The detection limits of the method for individual genotypes were examined using viral RNA extracted from 13 representative strains. The assay was also tested at 10 prefectural laboratories in Japan, designated as local reference laboratories for measles and rubella, to allow nationwide application of the assay. The detection limits and amplification efficiencies of the assay were similar among all the representative strains of the 13 genotypes. The TaqMan RT-PCR could detect approximately 90% of throat swab and urine samples taken up to 5days of illness. These samples were determined positive by a highly sensitive nested RT-PCR. The TaqMan RT-PCR could detect at least 10 pfu of rubella virus. Although the sensitivity was somewhat lower than that of the conventional nested RT-PCR, the TaqMan RT-PCR could be more practical to routine tests for rubella laboratory diagnosis and detection in view of the rapid response and reducing risks of contamination. Copyright © 2016 Elsevier B.V. All rights reserved.

Prevalence of Trichomonas vaginalis in Women Visiting 2 Obstetrics and Gynecology Clinics in Daegu, South Korea.

PubMed

Goo, Youn-Kyoung; Shin, Won-Sik; Yang, Hye-Won; Joo, So-Young; Song, Su-Min; Ryu, Jae-Sook; Lee, Won-Myung; Kong, Hyun-Hee; Lee, Won-Ki; Lee, Sang-Eun; Lee, Won-Ja; Chung, Dong-Il; Hong, Yeonchul

2016-02-01

This study explored epidemiological trends in trichomoniasis in Daegu, South Korea. Wet mount microscopy, PCR, and multiplex PCR were used to test for Trichomonas vaginalis in vaginal swab samples obtained from 621 women visiting 2 clinics in Daegu. Of the 621 women tested, microscopy detected T. vaginalis in 4 (0.6%) patients, PCR detected T. vaginalis in 19 (3.0%) patients, and multiplex PCR detected T. vaginalis in 12 (1.9%) patients. Testing via PCR demonstrated high sensitivity and high negative predictive value for T. vaginalis. Among the 19 women who tested positive for T. vaginalis according to PCR, 94.7% (18/19) reported vaginal signs and symptoms. Notably, more than 50% of T. vaginalis infections occurred in females younger than 30 years old, and 58% were unmarried. Multiplex PCR, which simultaneously detects pathogens from various sexually transmitted infections, revealed that 91.7% (11/12) of patients were infected with 2 or more pathogens. Mycoplasma hominis was the most prevalent co-infection pathogen with T. vaginalis, followed by Ureaplasma urealyticum and Chlamydia trachomatis. Our results indicate that PCR and multiplex PCR are the most sensitive tools for T. vaginalis diagnosis, rather than microscopy which has been routinely used to detect T. vaginalis infections in South Korea. Therefore, clinicians should take note of the high prevalence of T. vaginalis infections among adolescent and young women in order to prevent persistent infection and transmission of this disease.

A proof of concept study to assess the potential of PCR testing to detect natural Mycobacterium bovis infection in South American camelids

PubMed Central

2014-01-01

Background Cases of Mycobacterium bovis infection South American camelids have been increasing in Great Britain. Current antemortem immunological tests have some limitations. Cases at post mortem examination frequently show extensive pathology. The feasibility of detecting Mycobacterium bovis DNA in clinical samples was investigated. Findings A sensitive extraction methodology was developed and used on nasal swabs and faeces taken post-mortem to assess the potential for a PCR test to detect Mycobacterium bovis in clinical samples. The gross pathology of the studied South American camelids was scored and a significantly greater proportion of South American camelids with more severe pathology were positive in both the nasal swab and faecal PCR tests. A combination of the nasal swab and faecal PCR tests detected 63.9% of all the South American camelids with pathology that were tested. Conclusions The results suggest that antemortem diagnosis of Mycobacterium bovis in South American camelids may be possible using a PCR test on clinical samples, however more work is required to determine sensitivity and specificity, and the practicalities of applying the test in the field. PMID:24507471

Evaluation of Polymerase Chain Reaction for Detecting Coliform Bacteria in Drinking Water Sources.

PubMed

Isfahani, Bahram Nasr; Fazeli, Hossein; Babaie, Zeinab; Poursina, Farkhondeh; Moghim, Sharareh; Rouzbahani, Meisam

2017-01-01

Coliform bacteria are used as indicator organisms for detecting fecal pollution in water. Traditional methods including microbial culture tests in lactose-containing media and enzyme-based tests for the detection of β-galactosidase; however, these methods are time-consuming and less specific. The aim of this study was to evaluate polymerase chain reaction (PCR) for detecting coliform. Totally, 100 of water samples from Isfahan drinking water source were collected. Coliform bacteria and Escherichia coli were detected in drinking water using LacZ and LamB genes in PCR method performed in comparison with biochemical tests for all samples. Using phenotyping, 80 coliform isolates were found. The results of the biochemical tests illustrated 78.7% coliform bacteria and 21.2% E. coli . PCR results for LacZ and LamB genes were 67.5% and 17.5%, respectively. The PCR method was shown to be an effective, sensitive, and rapid method for detecting coliform and E. coli in drinking water from the Isfahan drinking water sources.

Improved detection of canine Angiostrongylus vasorum infection using real-time PCR and indirect ELISA.

PubMed

Jefferies, Ryan; Morgan, Eric R; Helm, Jenny; Robinson, Matthew; Shaw, Susan E

2011-12-01

This study reports the development of a real-time PCR assay and an indirect ELISA to improve on current detection of canine Angiostrongylus vasorum infection. A highly specific fluorescent probe-based, real-time PCR assay was developed to target the A. vasorum second internal transcribed spacer region and detected DNA in EDTA blood, lung tissue, broncho-alveolar larvage fluid, endotracheal mucus, pharyngeal swabs and faecal samples. PCR was fast (∼1 h), highly efficient when using EDTA blood samples, consistently detected a single molecule of parasite DNA and did not amplify DNA from other parasitic nematodes or definitive host species. An indirect ELISA was also developed using the soluble protein fraction from adult A. vasorum worms. Some cross-reactive antigen recognition was observed when tested against sera from dogs infected with Crenosoma vulpis (n = 8), Toxocara canis (n = 5) and Dirofilaria immitis (n = 5). This was largely overcome by setting the cut-off for a positive result at an appropriately high level. Field evaluation of the real-time PCR and ELISA was conducted by testing sera and EDTA blood from dogs with suspected A. vasorum infection (n = 148) and compared with the Baermann's larval migration test in faeces. Thirty-one dogs were positive by at least one test. Of these, 20 (65%) were detected by the Baermann method, 18 (58%) by blood PCR, 24 (77%) by ELISA and 28 (90%) by blood PCR and ELISA together. Combined testing using real-time PCR and ELISA therefore improved the detection rate of A. vasorum infection and holds promise for improved clinical diagnosis and epidemiological investigation.

Mycobacterium paratuberculosis detection in cow's milk in Argentina by immunomagnetic separation-PCR.

PubMed

Gilardoni, Liliana Rosa; Fernández, Bárbara; Morsella, Claudia; Mendez, Laura; Jar, Ana María; Paolicchi, Fernando Alberto; Mundo, Silvia Leonor

2016-01-01

The aim of this study was to standardize a diagnosis procedure to detect Mycobacterium avium subsp. paratuberculosis (Map) DNA in raw cow milk samples under field conditions. A procedure that combines both immunomagnetic separation and IS900-PCR detection (IMS-IS1 PCR) was employed on milk samples from 265 lactating Holstein cows from Map infected and uninfected herds in Argentina. IMS-IS1 PCR results were analyzed and compared with those obtained from milk and fecal culture and serum ELISA. The extent of agreement between both tests was determined by the Kappa test. IMS-IS1 PCR showed a detection limit of 10(1) CFU of Map/mL of milk, when 50:50 mix of monoclonal and polyclonal antibodies were used to coat magnetic beads. All of the 118 samples from the Map uninfected herds were negative for the set of the tests. In Map infected herds, 80 out of 147 cows tested positive by milk IMS-IS1 PCR (55%), of which 2 (1.4%) were also positive by milk culture, 15 (10%) by fecal culture, and 20 (14%) by serum ELISA. Kappa statistics (95% CI) showed a slight agreement between the different tests (<0.20), and the proportions of agreement were ≤0.55. The IMS-IS1 PCR method detected Map in milk of the cows that were not positive in other techniques. This is the first report dealing with the application of IMS-IS1 PCR in the detection of Map in raw milk samples under field conditions in Argentina. Copyright © 2016 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.

Simplified PCR for detection of Haemophilus ducreyi and diagnosis of chancroid.

PubMed Central

West, B; Wilson, S M; Changalucha, J; Patel, S; Mayaud, P; Ballard, R C; Mabey, D

1995-01-01

A simplified PCR was developed for detection of Haemophilus ducreyi in samples from chancroid patients. The strategy included a straightforward chloroform extraction sample preparation method, a one-tube nested PCR to minimize contamination risks, and a colorimetric method for detection of products. Primers were designed from published nucleotide sequences of the 16S rRNA gene of H. ducreyi, with longer outer primers for annealing at a higher temperature and shorter inner primers labelled with biotin and digoxigenin for binding with avidin and colorimetric detection. The PCR technique detected all 35 strains of H. ducreyi tested, from four different geographical regions, and was negative for other, related strains of bacteria and for the common contaminating bacteria tested. Of 25 samples from H. ducreyi culture-positive chancroid patients, 24 were PCR positive and 1 produced a weak reaction. Of 83 samples from clinical cases of chancroid in the Republic of South Africa, 69 were PCR positive. The sensitivity of PCR compared with that of clinical diagnosis was 83%. All 50 negative control samples were negative. Encouraging results were also obtained with a consecutive series of 25 genital ulcer patients in Tanzania, of whom 9 were PCR positive. The adaptations of this simplified PCR strategy, at the sensitivity and specificity levels obtained, mean it will be useful for detection of H. ducreyi in areas where the organism is endemic, particularly where testing by culture is difficult or impossible. PMID:7540625

Comparison of a PfHRP2-based rapid diagnostic test and PCR for malaria in a low prevalence setting in rural southern Zambia: implications for elimination.

PubMed

Laban, Natasha M; Kobayashi, Tamaki; Hamapumbu, Harry; Sullivan, David; Mharakurwa, Sungano; Thuma, Philip E; Shiff, Clive J; Moss, William J

2015-01-28

Rapid diagnostic tests (RDTs) detecting histidine-rich protein 2 (PfHRP2) antigen are used to identify individuals with Plasmodium falciparum infection even in low transmission settings seeking to achieve elimination. However, these RDTs lack sensitivity to detect low-density infections, produce false negatives for P. falciparum strains lacking pfhrp2 gene and do not detect species other than P. falciparum. Results of a PfHRP2-based RDT and Plasmodium nested PCR were compared in a region of declining malaria transmission in southern Zambia using samples from community-based, cross-sectional surveys from 2008 to 2012. Participants were tested with a PfHRP2-based RDT and a finger prick blood sample was spotted onto filter paper for PCR analysis and used to prepare blood smears for microscopy. Species-specific, real-time, quantitative PCR (q-PCR) was performed on samples that tested positive either by microscopy, RDT or nested PCR. Of 3,292 total participants enrolled, 12 (0.4%) tested positive by microscopy and 42 (1.3%) by RDT. Of 3,213 (98%) samples tested by nested PCR, 57 (1.8%) were positive, resulting in 87 participants positive by at least one of the three tests. Of these, 61 tested positive for P. falciparum by q-PCR with copy numbers ≤ 2 x 10(3) copies/μL, 5 were positive for both P. falciparum and Plasmodium malariae and 2 were positive for P. malariae alone. RDT detected 32 (53%) of P. falciparum positives, failing to detect three of the dual infections with P. malariae. Among 2,975 participants enrolled during a low transmission period between 2009 and 2012, sensitivity of the PfHRP2-based RDT compared to nested PCR was only 17%, with specificity of >99%. The pfhrp gene was detected in 80% of P. falciparum positives; however, comparison of copy number between RDT negative and RDT positive samples suggested that RDT negatives resulted from low parasitaemia and not pfhrp2 gene deletion. Low-density P. falciparum infections not identified by currently used PfHRP2-based RDTs and the inability to detect non-falciparum malaria will hinder progress to further reduce malaria in low transmission settings of Zambia. More sensitive and specific diagnostic tests will likely be necessary to identify parasite reservoirs and achieve malaria elimination.

Promising Nucleic Acid Lateral Flow Assay Plus PCR for Shiga Toxin-Producing Escherichia coli.

PubMed

Terao, Yoshitaka; Takeshita, Kana; Nishiyama, Yasutaka; Morishita, Naoki; Matsumoto, Takashi; Morimatsu, Fumiki

2015-08-01

Shiga toxin (Stx)-producing Escherichia coli (STEC) is a frequent cause of foodborne infections, and methods for rapid and reliable detection of STEC are needed. A nucleic acid lateral flow assay (NALFA) plus PCR was evaluated for detecting STEC after enrichment. When cell suspensions of 45 STEC strains, 14 non-STEC strains, and 13 non-E. coli strains were tested with the NALFA plus PCR, all of the STEC strains yielded positive results, and all of the non-STEC and non-E. coli strains yielded negative results. The lower detection limit for the STEC strains ranged from 0.1 to 1 pg of genomic DNA (about 20 to 200 CFU) per test, and the NALFA plus PCR was able to detect Stx1- and Stx2-producing E. coli strains with similar sensitivities. The ability of the NALFA plus PCR to detect STEC in enrichment cultures of radish sprouts, tomato, raw ground beef, and beef liver inoculated with 10-fold serially diluted STEC cultures was comparable to that of a real-time PCR assay (at a level of 100 to 100,000 CFU/ml in enrichment culture). The bacterial inoculation test in raw ground beef revealed that the lower detection limit of the NALFA plus PCR was also comparable to that obtained with a real-time PCR assay that followed the U.S. Department of Agriculture guidelines. Although further evaluation is required, these results suggest that the NALFA plus PCR is a specific and sensitive method for detecting STEC in a food manufacturing plant.

Fundamentals of Counting Statistics in Digital PCR: I Just Measured Two Target Copies-What Does It Mean?

PubMed

Tzonev, Svilen

2018-01-01

Current commercially available digital PCR (dPCR) systems and assays are capable of detecting individual target molecules with considerable reliability. As tests are developed and validated for use on clinical samples, the need to understand and develop robust statistical analysis routines increases. This chapter covers the fundamental processes and limitations of detecting and reporting on single molecule detection. We cover the basics of quantification of targets and sources of imprecision. We describe the basic test concepts: sensitivity, specificity, limit of blank, limit of detection, and limit of quantification in the context of dPCR. We provide basic guidelines how to determine those, how to choose and interpret the operating point, and what factors may influence overall test performance in practice.

Droplet digital PCR (ddPCR) vs quantitative real-time PCR (qPCR) approach for detection and quantification of Merkel cell polyomavirus (MCPyV) DNA in formalin fixed paraffin embedded (FFPE) cutaneous biopsies.

PubMed

Arvia, Rosaria; Sollai, Mauro; Pierucci, Federica; Urso, Carmelo; Massi, Daniela; Zakrzewska, Krystyna

2017-08-01

Merkel cell polyomavirus (MCPyV) is associated with Merkel cell carcinoma and high viral load in the skin was proposed as a risk factor for the occurrence of this tumour. MCPyV DNA was detected, with lower frequency, in different skin cancers but since the viral load was usually low, the real prevalence of viral DNA could be underestimated. To evaluate the performance of two assays (qPCR and ddPCR) for MCPyV detection and quantification in formalin fixed paraffin embedded (FFPE) tissue samples. Both assays were designed to simultaneous detection and quantification of both MCPyV as well as house-keeping DNA in clinical samples. The performance of MCPyV quantification was investigated using serial dilutions of cloned target DNA. We also evaluated the applicability of both tests for the analysis of 76 FFPE cutaneous biopsies. The two approaches resulted equivalent with regard to the reproducibility and repeatability and showed a high degree of linearity in the dynamic range tested in the present study. Moreover, qPCR was able to quantify ≥10 5 copies per reaction, while the upper limit of ddPCR was 10 4 copies. There was not significant difference between viral load measured by the two methods The detection limit of both tests was 0,15 copies per reaction, however, the number of positive samples obtained by ddPCR was higher than that obtained by qPCR (45% and 37% respectively). The ddPCR represents a better method for detection of MCPyV in FFPE biopsies, mostly these containing low copies number of viral genome. Copyright © 2017 Elsevier B.V. All rights reserved.

Usefulness of in-house PCR methods for hepatitis B virus DNA detection.

PubMed

Portilho, Moyra Machado; Baptista, Marcia Leite; da Silva, Messias; de Sousa, Paulo Sérgio Fonseca; Lewis-Ximenez, Lia Laura; Lampe, Elisabeth; Villar, Livia Melo

2015-10-01

The aim of the present study was to evaluate the performance of three in-house PCR techniques for HBV DNA detection and compare it with commercial quantitative methods to evaluate the usefulness of in-house methods for HBV diagnosis. Three panels of HBsAg reactive sera samples were evaluated: (i) 50 samples were examined using three methods for in-house qualitative PCR and the Cobas Amplicor HBV Monitor Assay; (ii) 87 samples were assayed using in-house semi-nested PCR and the Cobas TaqMan HBV test; (iii) 11 serial samples obtained from 2 HBV-infected individuals were assayed using the Cobas Amplicor HBV test and semi-nested PCR. In panel I, HBV DNA was detected in 44 samples using the Cobas Amplicor HBV test, 42 samples using semi-nested PCR (90% concordance with Cobas Amplicor), 22 samples using PCR for the core gene (63.6% concordance) and 29 samples using single-round PCR for the pre-S/S gene (75% concordance). In panel II, HBV DNA was quantified in 78 of the 87 HBsAg reactive samples using Cobas TaqMan but 52 samples using semi-nested PCR (67.8% concordance). HBV DNA was detected in serial samples until the 17th and 26th week after first donation using in-house semi-nested PCR and the Cobas Amplicor HBV test, respectively. In-house semi-nested PCR presented adequate concordance with commercial methods as an alternative method for HBV molecular diagnosis in low-resource settings. Copyright © 2015 Elsevier B.V. All rights reserved.

Concordance of polymerase chain reaction with human immunodeficiency virus antibody detection.

PubMed

Horsburgh, C R; Ou, C Y; Jason, J; Holmberg, S D; Lifson, A R; Moore, J L; Ward, J W; Seage, G R; Mayer, K H; Evatt, B L

1990-08-01

To evaluate the correlation of detection of human immunodeficiency virus (HIV) by polymerase chain reaction (PCR) with detection of HIV antibody, 271 simultaneous serum and peripheral blood mononuclear cell samples were examined from 242 persons whose activities placed them at increased risk for HIV infection: 142 from homosexual men, 86 from hemophilic men, and 43 from heterosexual partners of HIV-infected persons. PCR was performed using the gag region primer pair SK38/39 and the env region primer pairs SK68/69 and CO71/72. Amplified HIV DNA was detected using specific oligomer probes. Of 63 HIV antibody-positive samples, 58 (92%) had HIV DNA by PCR. Of 208 HIV antibody-negative samples, 7 (3.4%) had HIV DNA by PCR. On follow-up, 4 of the latter persons were seropositive when next tested; 2 were well and antibody- and PCR-negative; 1 had died of a stroke before retesting. Thus, PCR detects HIV in most antibody-positive persons; detection is increased by use of multiple primer pairs. PCR-positive antibody-negative specimens may indicate HIV infection in which antibody has not yet developed or may be false-positive PCR results. When PCR is discordant with HIV antibody, testing of additional specimens and clinical follow-up are necessary to assess HIV infection status.

Comparison of the clinical performances of the AdvanSure HPV Screening Real-Time PCR, the Abbott Real-Time High-Risk HPV Test, and the Hybrid Capture High-Risk HPV DNA Test for Cervical Cancer Screening.

PubMed

Chung, Hae-Sun; Hahm, Chorong; Lee, Miae

2014-09-01

The clinical performance of three human papillomavirus (HPV) DNA commercial assays for cervical cancer screening was evaluated; the AdvanSure HPV Screening Real-Time PCR (AdvanSure PCR; LG Life Sciences) that was developed recently for the detection of both high-risk and low-risk genotypes, the Abbott RealTime High-Risk HPV Test (Abbott PCR; Abbott Molecular) and the Hybrid Capture High-Risk HPV DNA test (HC2; Qiagen). The three different HPV DNA tests were compared using cytology samples obtained from 619 women who underwent routine cervical cancer screening. The gold-standard assay was histopathological confirmation of cervical intraepithelial neoplasia of grade 2 or worse. The clinical sensitivities of the AdvanSure PCR, the Abbott PCR and the HC2 for the detection of cervical intraepithelial neoplasia of grade 2 or worse were 95.5%, 95.5% and 100%, respectively, while the clinical specificities were 61.6%, 86.4% and 83.3%, respectively. There were no significant differences in the clinical sensitivities of the Abbott PCR and the AdvanSure PCR compared to the HC2. The clinical specificities of the Abbott PCR and the AdvanSure PCR for the detection of HPV types 16/18 were 97.8% and 98.5%, respectively. For cervical cancer screening, all three tests showed relatively good clinical sensitivities, but the AdvanSure PCR had lower clinical specificity than the Abbott PCR and the HC2. The AdvanSure PCR and the Abbott PCR assays have the advantage of being automated and the ability to distinguish between HPV types 16/18 and other HPV types. The two real-time PCR assays could be useful tools in HPV testing for cervical cancer screening. Copyright © 2014 Elsevier B.V. All rights reserved.

Molecular diagnosis of Legionella infections--Clinical utility of front-line screening as part of a pneumonia diagnostic algorithm.

PubMed

Gadsby, Naomi J; Helgason, Kristjan O; Dickson, Elizabeth M; Mills, Jonathan M; Lindsay, Diane S J; Edwards, Giles F; Hanson, Mary F; Templeton, Kate E

2016-02-01

Urinary antigen testing for Legionella pneumophila serogroup 1 is the leading rapid diagnostic test for Legionnaires' Disease (LD); however other Legionella species and serogroups can also cause LD. The aim was to determine the utility of front-line L. pneumophila and Legionella species PCR in a severe respiratory infection algorithm. L. pneumophila and Legionella species duplex real-time PCR was carried out on 1944 specimens from hospitalised patients over a 4 year period in Edinburgh, UK. L. pneumophila was detected by PCR in 49 (2.7%) specimens from 36 patients. During a LD outbreak, combined L. pneumophila respiratory PCR and urinary antigen testing had optimal sensitivity and specificity (92.6% and 98.3% respectively) for the detection of confirmed cases. Legionella species was detected by PCR in 16 (0.9%) specimens from 10 patients. The 5 confirmed and 1 probable cases of Legionella longbeachae LD were both PCR and antibody positive. Front-line L. pneumophila and Legionella species PCR is a valuable addition to urinary antigen testing as part of a well-defined algorithm. Cases of LD due to L. longbeachae might be considered laboratory-confirmed when there is a positive Legionella species PCR result and detection of L. longbeachae specific antibody response. Copyright © 2015 The British Infection Association. Published by Elsevier Ltd. All rights reserved.

Evaluation of culture- and PCR-based detection methods for Escherichia coli O157:H7 in inoculated ground beeft.

PubMed

Arthur, Terrance M; Bosilevac, Joseph M; Nou, Xiangwu; Koohmaraie, Mohammad

2005-08-01

Currently, several beef processors employ test-and-hold systems for increased quality control of ground beef. In such programs, each lot of product must be tested and found negative for Escherichia coli O157:H7 prior to release of the product into commerce. Optimization of three testing attributes (detection time, specificity, and sensitivity) is critical to the success of such strategies. Because ground beef is a highly perishable product, the testing methodology used must be as rapid as possible. The test also must have a low false-positive result rate so product is not needlessly discarded. False-negative results cannot be tolerated because they would allow contaminated product to be released and potentially cause disease. In this study, two culture-based and three PCR-based methods for detecting E. coli O157:H7 in ground beef were compared for their abilities to meet the above criteria. Ground beef samples were individually spiked with five genetically distinct strains of E. coli O157: H7 at concentrations of 17 and 1.7 CFU/65 g and then subjected to the various testing methodologies. There was no difference (P > 0.05) in the abilities of the PCR-based methods to detect E. coli O157:H7 inoculated in ground beef at 1.7 CFU/65 g. The culture-based systems detected more positive samples than did the PCR-based systems, but the detection times (21 to 48 h) were at least 9 h longer than those for the PCR-based methods (7.5 to 12 h). Ground beef samples were also spiked with potentially cross-reactive strains. The PCR-based systems that employed an immunomagnetic separation step prior to detection produced fewer false-positive results.

A cost effective real-time PCR for the detection of adenovirus from viral swabs

PubMed Central

2013-01-01

Compared to traditional testing strategies, nucleic acid amplification tests such as real-time PCR offer many advantages for the detection of human adenoviruses. However, commercial assays are expensive and cost prohibitive for many clinical laboratories. To overcome fiscal challenges, a cost effective strategy was developed using a combination of homogenization and heat treatment with an “in-house” real-time PCR. In 196 swabs submitted for adenovirus detection, this crude extraction method showed performance characteristics equivalent to viral DNA obtained from a commercial nucleic acid extraction. In addition, the in-house real-time PCR outperformed traditional testing strategies using virus culture, with sensitivities of 100% and 69.2%, respectively. Overall, the combination of homogenization and heat treatment with a sensitive in-house real-time PCR provides accurate results at a cost comparable to viral culture. PMID:23758993

Detection of Mycobacterium bovis in Bovine and Bubaline Tissues Using Nested-PCR for TbD1

PubMed Central

Araújo, Cristina P.; Osório, Ana Luiza A. R.; Jorge, Kláudia S. G.; Ramos, Carlos Alberto N.; Filho, Antonio Francisco S.; Vidal, Carlos Eugênio S.; Roxo, Eliana; Nishibe, Christiane; Almeida, Nalvo F.; Júnior, Antônio A. F.; Silva, Marcio R.; Neto, José Diomedes B.; Cerqueira, Valíria D.; Zumárraga, Martín J.; Araújo, Flábio R.

2014-01-01

In the present study, a nested-PCR system, targeting the TbD1 region, involving the performance of conventional PCR followed by real-time PCR, was developed to detect Mycobacterium bovis in bovine/bubaline tissue homogenates. The sensitivity and specificity of the reactions were assessed with DNA samples extracted from tuberculous and non-tuberculous mycobacteria, as well as other actinomycetales species and DNA samples extracted directly from bovine and bubaline tissue homogenates. In terms of analytical sensitivity, the DNA of M. bovis AN5 was detected up to 1.56 ng with conventional PCR, 97.6 pg with real-time PCR, and 1.53 pg with nested-PCR in the reaction mixture. The nested-PCR exhibited 100% analytical specificity for M. bovis when tested with the DNA of reference strains of environmental mycobacteria and closely-related Actinomycetales. A clinical sensitivity value of 76.0% was detected with tissue samples from animals that exhibited positive results in the comparative intradermal tuberculin test (CITT), as well as from those with lesions compatible with tuberculosis (LCT) that rendered positive cultures. A clinical specificity value of 100% was detected with tissue samples from animals with CITT- results, with no visible lesions (NVL) and negative cultures. No significant differences were found between the nested-PCR and culture in terms of detecting CITT+ animals with LCT or with NVL. No significant differences were recorded in the detection of CITT- animals with NVL. However, nested-PCR detected a significantly higher number of positive animals than the culture in the group of animals exhibiting LCT with no previous records of CITT. The use of the nested-PCR assay to detect M. bovis in tissue homogenates provided a rapid diagnosis of bovine and bubaline tuberculosis. PMID:24618787

Detection of Mycobacterium bovis in bovine and bubaline tissues using nested-PCR for TbD1.

PubMed

Araújo, Cristina P; Osório, Ana Luiza A R; Jorge, Kláudia S G; Ramos, Carlos Alberto N; Filho, Antonio Francisco S; Vidal, Carlos Eugênio S; Roxo, Eliana; Nishibe, Christiane; Almeida, Nalvo F; Júnior, Antônio A F; Silva, Marcio R; Neto, José Diomedes B; Cerqueira, Valíria D; Zumárraga, Martín J; Araújo, Flábio R

2014-01-01

In the present study, a nested-PCR system, targeting the TbD1 region, involving the performance of conventional PCR followed by real-time PCR, was developed to detect Mycobacterium bovis in bovine/bubaline tissue homogenates. The sensitivity and specificity of the reactions were assessed with DNA samples extracted from tuberculous and non-tuberculous mycobacteria, as well as other actinomycetales species and DNA samples extracted directly from bovine and bubaline tissue homogenates. In terms of analytical sensitivity, the DNA of M. bovis AN5 was detected up to 1.56 ng with conventional PCR, 97.6 pg with real-time PCR, and 1.53 pg with nested-PCR in the reaction mixture. The nested-PCR exhibited 100% analytical specificity for M. bovis when tested with the DNA of reference strains of environmental mycobacteria and closely-related Actinomycetales. A clinical sensitivity value of 76.0% was detected with tissue samples from animals that exhibited positive results in the comparative intradermal tuberculin test (CITT), as well as from those with lesions compatible with tuberculosis (LCT) that rendered positive cultures. A clinical specificity value of 100% was detected with tissue samples from animals with CITT- results, with no visible lesions (NVL) and negative cultures. No significant differences were found between the nested-PCR and culture in terms of detecting CITT+ animals with LCT or with NVL. No significant differences were recorded in the detection of CITT- animals with NVL. However, nested-PCR detected a significantly higher number of positive animals than the culture in the group of animals exhibiting LCT with no previous records of CITT. The use of the nested-PCR assay to detect M. bovis in tissue homogenates provided a rapid diagnosis of bovine and bubaline tuberculosis.

Detecting Malaria Hotspots: A Comparison of Rapid Diagnostic Test, Microscopy, and Polymerase Chain Reaction

PubMed Central

Mogeni, Polycarp; Williams, Thomas N; Omedo, Irene; Kimani, Domtila; Ngoi, Joyce M; Mwacharo, Jedida; Morter, Richard; Nyundo, Christopher; Wambua, Juliana; Nyangweso, George; Kapulu, Melissa; Fegan, Gregory; Bejon, Philip

2017-01-01

Abstract Background Malaria control strategies need to respond to geographical hotspots of transmission. Detection of hotspots depends on the sensitivity of the diagnostic tool used. Methods We conducted cross-sectional surveys in 3 sites within Kilifi County, Kenya, that had variable transmission intensities. Rapid diagnostic test (RDT), microscopy, and polymerase chain reaction (PCR) were used to detect asymptomatic parasitemia, and hotspots were detected using the spatial scan statistic. Results Eight thousand five hundred eighty-one study participants were surveyed in 3 sites. There were statistically significant malaria hotspots by RDT, microscopy, and PCR for all sites except by microscopy in 1 low transmission site. Pooled data analysis of hotspots by PCR overlapped with hotspots by microscopy at a moderate setting but not at 2 lower transmission settings. However, variations in degree of overlap were noted when data were analyzed by year. Hotspots by RDT were predictive of PCR/microscopy at the moderate setting, but not at the 2 low transmission settings. We observed long-term stability of hotspots by PCR and microscopy but not RDT. Conclusion Malaria control programs may consider PCR testing to guide asymptomatic malaria hotspot detection once the prevalence of infection falls. PMID:28973672

A simple nucleic acid hybridization/latex agglutination assay for the rapid detection of polymerase chain reaction amplicons.

PubMed

Vollenhofer-Schrumpf, Sabine; Buresch, Ronald; Schinkinger, Manfred

2007-03-01

We have developed a new method for the detection of nucleic acid hybridization, based on a simple latex agglutination test that can be evaluated by the unaided eye. Nucleic acid, e.g., a polymerase chain reaction (PCR) product, is denatured and incubated with polystyrene beads carrying covalently bound complementary oligonucleotide sequences. Hybridization of the nucleic acids leads to aggregation of the latex particles, thereby verifying the presence of target sequence. The test is performed at room temperature, and results are available within 10 min. As a proof of principle, the hybridization/latex agglutination assay was applied to the detection of purified PCR fragments either specific for Salmonella spp. or a synthetic sequence, and to the detection of Salmonella enterica in artificially contaminated chicken samples. A few nanograms of purified PCR fragments were detectable. In artificially contaminated chicken samples, 3 colony-forming units (cfu)/25 g were detected in one of three replicates, and 30 cfu/25 g were detected in both of two replicates when samples for PCR were taken directly from primary enrichment, demonstrating the practical applicability of this test system. Even multiplex detection might be achievable. This novel kind of assay could be useful for a range of applications where hybridization of nucleic acids, e.g., PCR fragments, is to be detected.

Quantitative Detection and Genotyping of Helicobacter pylori from Stool using Droplet Digital PCR Reveals Variation in Bacterial Loads that Correlates with cagA Virulence Gene Carriage.

PubMed

Talarico, Sarah; Safaeian, Mahboobeh; Gonzalez, Paula; Hildesheim, Allan; Herrero, Rolando; Porras, Carolina; Cortes, Bernal; Larson, Ann; Fang, Ferric C; Salama, Nina R

2016-08-01

Epidemiologic studies of the carcinogenic stomach bacterium Helicobacter pylori have been limited by the lack of noninvasive detection and genotyping methods. We developed a new stool-based method for detection, quantification, and partial genotyping of H. pylori using droplet digital PCR (ddPCR), which allows for increased sensitivity and absolute quantification by PCR partitioning. Stool-based ddPCR assays for H. pylori 16S gene detection and cagA virulence gene typing were tested using a collection of 50 matched stool and serum samples from Costa Rican volunteers and 29 H. pylori stool antigen-tested stool samples collected at a US hospital. The stool-based H. pylori 16S ddPCR assay had a sensitivity of 84% and 100% and a specificity of 100% and 71% compared to serology and stool antigen tests, respectively. The stool-based cagA genotyping assay detected cagA in 22 (88%) of 25 stools from CagA antibody-positive individuals and four (16%) of 25 stools from CagA antibody-negative individuals from Costa Rica. All 26 of these samples had a Western-type cagA allele. Presence of serum CagA antibodies was correlated with a significantly higher load of H. pylori in the stool. The stool-based ddPCR assays are a sensitive, noninvasive method for detection, quantification, and partial genotyping of H. pylori. The quantitative nature of ddPCR-based H. pylori detection revealed significant variation in bacterial load among individuals that correlates with presence of the cagA virulence gene. These stool-based ddPCR assays will facilitate future population-based epidemiologic studies of this important human pathogen. © 2015 John Wiley & Sons Ltd.

Detection of respiratory viruses and bacteria in children using a twenty-two target reverse-transcription real-time PCR (RT-qPCR) panel.

PubMed

Ellis, Chelsey; Misir, Amita; Hui, Charles; Jabbour, Mona; Barrowman, Nicholas; Langill, Jonathan; Bowes, Jennifer; Slinger, Robert

2016-05-01

Rapid detection of the wide range of viruses and bacteria that cause respiratory infection in children is important for patient care and antibiotic stewardship. We therefore designed and evaluated a ready-to-use 22 target respiratory infection reverse-transcription real-time polymerase chain reaction (RT-qPCR) panel to determine if this would improve detection of these agents at our pediatric hospital. RT-qPCR assays for twenty-two target organisms were dried-down in individual wells of 96 well plates and saved at room temperature. Targets included 18 respiratory viruses and 4 bacteria. After automated nucleic acid extraction of nasopharyngeal aspirate (NPA) samples, rapid qPCR was performed. RT-qPCR results were compared with those obtained by the testing methods used at our hospital laboratories. One hundred fifty-nine pediatric NPA samples were tested with the RT-qPCR panel. One or more respiratory pathogens were detected in 132/159 (83%) samples. This was significantly higher than the detection rate of standard methods (94/159, 59%) (P<0.001). This difference was mainly due to improved RT-qPCR detection of rhinoviruses, parainfluenza viruses, bocavirus, and coronaviruses. The panel internal control assay performance remained stable at room temperature storage over a two-month testing period. The RT-qPCR panel was able to identify pathogens in a high proportion of respiratory samples. The panel detected more positive specimens than the methods in use at our hospital. The pre-made panel format was easy to use and rapid, with results available in approximately 90 minutes. We now plan to determine if use of this panel improves patient care and antibiotic stewardship.

Droplet digital polymerase chain reaction (PCR) outperforms real-time PCR in the detection of environmental DNA from an invasive fish species.

PubMed

Doi, Hideyuki; Takahara, Teruhiko; Minamoto, Toshifumi; Matsuhashi, Saeko; Uchii, Kimiko; Yamanaka, Hiroki

2015-05-05

Environmental DNA (eDNA) has been used to investigate species distributions in aquatic ecosystems. Most of these studies use real-time polymerase chain reaction (PCR) to detect eDNA in water; however, PCR amplification is often inhibited by the presence of organic and inorganic matter. In droplet digital PCR (ddPCR), the sample is partitioned into thousands of nanoliter droplets, and PCR inhibition may be reduced by the detection of the end-point of PCR amplification in each droplet, independent of the amplification efficiency. In addition, real-time PCR reagents can affect PCR amplification and consequently alter detection rates. We compared the effectiveness of ddPCR and real-time PCR using two different PCR reagents for the detection of the eDNA from invasive bluegill sunfish, Lepomis macrochirus, in ponds. We found that ddPCR had higher detection rates of bluegill eDNA in pond water than real-time PCR with either of the PCR reagents, especially at low DNA concentrations. Limits of DNA detection, which were tested by spiking the bluegill DNA to DNA extracts from the ponds containing natural inhibitors, found that ddPCR had higher detection rate than real-time PCR. Our results suggest that ddPCR is more resistant to the presence of PCR inhibitors in field samples than real-time PCR. Thus, ddPCR outperforms real-time PCR methods for detecting eDNA to document species distributions in natural habitats, especially in habitats with high concentrations of PCR inhibitors.

Monitoring food pathogens: Novel instrumentation for cassette PCR testing

PubMed Central

Hunt, Darin; Figley, Curtis; Lauzon, Jana; Figley, Rachel; Pilarski, Linda M.; McMullen, Lynn M.; Pilarski, Patrick M.

2018-01-01

In this manuscript, we report the design and development of a fast, reliable instrument to run gel-based cassette polymerase chain reactions (PCR). Here termed the GelCycler Mark II, our instrument is a miniaturized molecular testing system that is fast, low cost and sensitive. Cassette PCR utilizes capillary reaction units that carry all reagents needed for PCR, including primers and Taq polymerase, except the sample, which is loaded at the time of testing. Cassette PCR carries out real time quantitative PCR followed by melt curve analysis (MCA) to verify amplicon identity at the expected melt temperature (Tm). The cassette PCR technology is well developed, particularly for detecting pathogens, and has been rigorously validated for detecting pathogenic Escherichia coli in meat samples. However, the work has been hindered by the lack of a robust and stable instrument to carry out the PCR, which requires fast and accurate temperature regulation, improved light delivery and fluorescent recording, and faster PCR reactions that maintain a high sensitivity of detection. Here, we report design and testing of a new instrument to address these shortcomings and to enable standardized testing by cassette PCR and commercial manufacture of a robust and accurate instrument that can be mass produced to deliver consistent performance. As a corollary to our new instrument development, we also report the use of an improved design approach using a machined aluminum cassette to meet the new instrument standards, prevent any light bleed across different trenches in each cassette, and allow testing of a larger number of samples for more targets in a single run. The GelCycler Mark II can detect and report E. coli contamination in 41 minutes. Sample positives are defined in as having a melt curve comparable to the internal positive control, with peak height exceeding that of the internal negative control. In a fractional analysis, as little as 1 bacterium per capillary reaction unit is directly detectable, with no enrichment step, in 35 cycles of PCR/MCA, in a total time of 53 minutes, making this instrument and technology among the very best for speed and sensitivity in screening food for pathogenic contamination. PMID:29746561

Use of polymerase chain reaction technique to confirm VecTest screening results in Plasmodium falciparum and Plasmodium vivax VK 210 laboratory-infected Anopheles stephensi mosquitoes.

PubMed

Santos-Ciminera, Patricia D; Acheé, Nicole L; Quinnan, Gerald V; Roberts, Donald R

2004-09-01

We evaluated polymerase chain reaction (PCR) to confirm immunoassays for malaria parasites in mosquito pools after a failure to detect malaria with PCR during an outbreak in which pools tested positive using VecTest and enzyme-linked immunosorbent assay (ELISA). We combined VecTest, ELISA, and PCR to detect Plasmodium falciparum and Plasmodium vivax VK 210. Each mosquito pool, prepared in triplicate, consisted of 1 exposed Anopheles stephensi and up to 9 unfed mosquitoes. The results of VecTest and ELISA were concordant. DNA from a subset of the pools, 1 representative of each ratio of infected to uninfected mosquitoes, was extracted and used as template in PCR. All P. vivax pools were PCR positive but some needed additional processing for removal of apparent inhibitors before positive results were obtained. One of the pools selected for P. falciparum was negative by PCR, probably because of losses or contamination during DNA extraction; 2 remaining pools at this ratio were PCR positive. Testing pools by VecTest, ELISA, and PCR is feasible, and PCR is useful for confirmation of immunoassays. An additional step might be needed to remove potential inhibitors from pools prior to PCR.

Evaluation of Polymerase Chain Reaction for Detecting Coliform Bacteria in Drinking Water Sources

PubMed Central

Isfahani, Bahram Nasr; Fazeli, Hossein; Babaie, Zeinab; Poursina, Farkhondeh; Moghim, Sharareh; Rouzbahani, Meisam

2017-01-01

Background: Coliform bacteria are used as indicator organisms for detecting fecal pollution in water. Traditional methods including microbial culture tests in lactose-containing media and enzyme-based tests for the detection of β-galactosidase; however, these methods are time-consuming and less specific. The aim of this study was to evaluate polymerase chain reaction (PCR) for detecting coliform. Materials and Methods: Totally, 100 of water samples from Isfahan drinking water source were collected. Coliform bacteria and Escherichia coli were detected in drinking water using LacZ and LamB genes in PCR method performed in comparison with biochemical tests for all samples. Results: Using phenotyping, 80 coliform isolates were found. The results of the biochemical tests illustrated 78.7% coliform bacteria and 21.2% E. coli. PCR results for LacZ and LamB genes were 67.5% and 17.5%, respectively. Conclusion: The PCR method was shown to be an effective, sensitive, and rapid method for detecting coliform and E. coli in drinking water from the Isfahan drinking water sources. PMID:29142893

Role of PCR method using IS6110 primer in detecting Mycobacterium tuberculosis among the clinically diagnosed childhood tuberculosis patients at an urban hospital in Dhaka, Bangladesh.

PubMed

Kabir, Senjuti; Uddin, Mohammad Khaja Mafij; Chisti, Mohammod Jobayer; Fannana, Tilka; Haque, Mohammad Enamul; Uddin, Muhammad Reaj; Banu, Sayera; Ahmed, Tahmeed

2018-03-01

Better methods are needed for the accurate detection of child tuberculosis (TB). This study compared different laboratory tests and evaluated IS6110 PCR for the detection of Mycobacterium tuberculosis (MTB) among clinically diagnosed child TB patients. A total of 102 paediatric patients (<15 years old) with clinically diagnosed TB were enrolled in this study. The patients were admitted to the icddr,b hospital in Dhaka between 2003 and 2005. Sputum/gastric lavage samples were collected for smear microscopy, culture (solid/Lowenstein-Jensen medium and liquid/MGIT), and IS6110 PCR testing. The sensitivity, specificity, and positive and negative predictive values (PPV, NPV) of smear microscopy and PCR were compared to the two culture methods. Three patients were positive on smear microscopy (2.9%). MTB was detected by conventional culture in 15.7% (16/102), liquid culture in 14% (14/100), and IS6110 PCR in 61.8% (63/102). PCR detected an additional 45 patients who were undetected with the three other tests. Compared to conventional and liquid culture, respectively, smear microscopy showed sensitivity of 18.8% and 21.4%, specificity of 100% individually, PPV of 100% individually, and NPV of 86.9% and 88.7%, whereas PCR had sensitivity of 87.5% and 92.9%, specificity of 43% individually, PPV of 22.2% and 21%, and NPV of 94.9% and 97.4%. PCR can be useful compared to smear microscopy and culture methods and is applicable as a rapid screening test for child TB. A larger scale study is required to determine its diagnostic efficacy in improving the detection of child TB in the presence and absence of severe malnutrition. Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.

[Detection of KRAS mutation in colorectal cancer patients' cfDNA with droplet digital PCR].

PubMed

Luo, Yuwen; Li, Yao

2018-03-25

This study aims to develop a new method for the detection of KRAS mutations related to colorectal cancer in cfDNA, and to evaluate the sensitivity and accuracy of the detection. We designed a method of cfDNA based KRAS detection by droplets digital PCR (ddPCR). The theoretical performance of the method is evaluated by reference standard and compared to the ARMS PCR method. Two methods, ddPCR and qPCR, were successfully established to detect KRAS wild type and 7 mutants. Both methods were validated using plasmid standards and actual samples. The results were evaluated by false positive rate, linearity, and limit of detection. Finally, 52 plasma cfDNA samples from patients and 20 samples from healthy people were tested, the clinical sensitivity is 97.64%, clinical specificity is 81.43%. ddPCR method shows higher performance than qPCR. The LOD of ddPCR method reached single digits of cfDNA copies, it can detect as low as 0.01% to 0.04% mutation abundance.

Novel PCR Assays Complement Laser Biosensor-Based Method and Facilitate Listeria Species Detection from Food.

PubMed

Kim, Kwang-Pyo; Singh, Atul K; Bai, Xingjian; Leprun, Lena; Bhunia, Arun K

2015-09-08

The goal of this study was to develop the Listeria species-specific PCR assays based on a house-keeping gene (lmo1634) encoding alcohol acetaldehyde dehydrogenase (Aad), previously designated as Listeria adhesion protein (LAP), and compare results with a label-free light scattering sensor, BARDOT (bacterial rapid detection using optical scattering technology). PCR primer sets targeting the lap genes from the species of Listeria sensu stricto were designed and tested with 47 Listeria and 8 non-Listeria strains. The resulting PCR primer sets detected either all species of Listeria sensu stricto or individual L. innocua, L. ivanovii and L. seeligeri, L. welshimeri, and L. marthii without producing any amplified products from other bacteria tested. The PCR assays with Listeria sensu stricto-specific primers also successfully detected all species of Listeria sensu stricto and/or Listeria innocua from mixed culture-inoculated food samples, and each bacterium in food was verified by using the light scattering sensor that generated unique scatter signature for each species of Listeria tested. The PCR assays based on the house-keeping gene aad (lap) can be used for detection of either all species of Listeria sensu stricto or certain individual Listeria species in a mixture from food with a detection limit of about 10⁴ CFU/mL.

Novel PCR Assays Complement Laser Biosensor-Based Method and Facilitate Listeria Species Detection from Food

PubMed Central

Kim, Kwang-Pyo; Singh, Atul K.; Bai, Xingjian; Leprun, Lena; Bhunia, Arun K.

2015-01-01

The goal of this study was to develop the Listeria species-specific PCR assays based on a house-keeping gene (lmo1634) encoding alcohol acetaldehyde dehydrogenase (Aad), previously designated as Listeria adhesion protein (LAP), and compare results with a label-free light scattering sensor, BARDOT (bacterial rapid detection using optical scattering technology). PCR primer sets targeting the lap genes from the species of Listeria sensu stricto were designed and tested with 47 Listeria and 8 non-Listeria strains. The resulting PCR primer sets detected either all species of Listeria sensu stricto or individual L. innocua, L. ivanovii and L. seeligeri, L. welshimeri, and L. marthii without producing any amplified products from other bacteria tested. The PCR assays with Listeria sensu stricto-specific primers also successfully detected all species of Listeria sensu stricto and/or Listeria innocua from mixed culture-inoculated food samples, and each bacterium in food was verified by using the light scattering sensor that generated unique scatter signature for each species of Listeria tested. The PCR assays based on the house-keeping gene aad (lap) can be used for detection of either all species of Listeria sensu stricto or certain individual Listeria species in a mixture from food with a detection limit of about 104 CFU/mL. PMID:26371000

Development of an In-House Multiplex Nested RT-PCR Method for Detecting Acute HIV-1 Infection in High Risk Populations.

PubMed

Liu, Zhiying; Li, Wei; Xu, Meng; Sheng, Bo; Yang, Zixuan; Jiao, Yanmei; Zhang, Tong; Mou, Danlei; Chen, Dexi; Wu, Hao

2015-01-01

The detection of acute HIV infection (AHI) among high risk populations can help reduce secondary transmission of HIV. The nucleic acid testing (NAT) can shorten the test window period by up to 7-12 days. In this study, we describe an in-house NAT based on the multiplex nested RT-PCR method to detect the HIV RNA. We also evaluated it in a high risk cohort in Beijing. Four primer pairs were designed and evaluated for the detection of different HIV-1 subtypes in group M. Multiplex RT-PCR and nested PCR were performed. The sensitivity, specialty, primers compatibility among HIV subtypes were evaluated simultaneously. In an MSM cohort in Beijing during a 3-year period, a total of 11,808 blood samples that were negative by ELISA or indeterminate by Western blot were analyzed by this multiplex nested RT-PCR with pooling strategy. The multiplex nested RT-PCR was successfully applied for the detection of at least six HIV-1 subtypes. The sensitivity was 40 copies/ml and the specificity was 100%. A total of 29 people were tested HIV-1 positive with acute infection in a MSM cohort of Beijing during a 3 years period. This multiplex nested RT-PCR provides a useful tool for the rapid detection of acute HIV-1 infection. When used in combination with the 3(rd) generation ELISA, it can improve the detection rate of HIV infection, especially in the source limited regions.

Development of an improved PCR-ICT hybrid assay for direct detection of Legionellae and Legionella pneumophila from cooling tower water specimens.

PubMed

Horng, Yu-Tze; Soo, Po-Chi; Shen, Bin-Jon; Hung, Yu-Li; Lo, Kai-Yin; Su, Hsun-Pi; Wei, Jun-Rong; Hsieh, Shang-Chen; Hsueh, Po-Ren; Lai, Hsin-Chih

2006-06-01

A novelly improved polymerase chian reaction and immunochromatography test (PCR-ICT) hybrid assay comprising traditional multiplex-nested PCR and ICT, (a lateral-flow device) was developed for direct detection of Legionella bacteria from environmental cooling tower samples. The partial 16S rDNA (specific for Legionella spp.) and dnaJ (specific for Legionella pneumophila) genes from Legionella chromosome were first specifically amplified by multiplex-nested PCR, respectively, followed by detection using ICT strip. Reading of results was based on presence or absence of the two test lines on the strips. Presence of test line 1 indicated existence of Legionella spp. specific 16S rDNA and identified Legionella spp. Presence of test line 2 further indicated existence of dnaJ and thus specifically identified L. pneumophila. In contrast, for non-Legionellae bacteria no test line formation was observed. Results of direct detection of Legionella bacteria and L. pneumophila from water tower specimens by this assay showed 100% sensitivity, and 96.6% and 100% specificity, respectively compared with traditional culture, biochemical and serological identification methods. The PCR-ICT hybrid assay does not require sophisticated equipment and was proved to be practically useful in rapid and direct Legionellae detection from environmental water samples.

Rapid and sensitive detection of Zika virus by reverse transcription loop-mediated isothermal amplification.

PubMed

Wang, Xuan; Yin, Fenggui; Bi, Yuhai; Cheng, Gong; Li, Jing; Hou, Lidan; Li, Yunlong; Yang, Baozhi; Liu, Wenjun; Yang, Limin

2016-12-01

Zika virus (ZIKV) is an arbovirus that recently emerged and has expanded worldwide, causing a global threat and raising international concerns. Current molecular diagnostics, e.g., real-time PCR and reverse transcription PCR (RT-PCR), are time consuming, expensive, and can only be deployed in a laboratory instead of for field diagnostics. This study aimed to develop a one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) platform showing sensitivity, specificity, and more convenience than previous methods, being easily distributed and implemented. Specific primers were designed and screened to target the entire ZIKV genome. The analytical sensitivity and specificity of the assay were evaluated and compared with traditional PCR and quantitative real-time PCR. Three different simulated clinical sample quick preparation protocols were evaluated to establish a rapid and straightforward treatment procedure for clinical specimens in open field detection. The RT-LAMP assay for detection of ZIKV demonstrated superior specificity and sensitivity compared to traditional PCR at the optimum reaction temperature. For the ZIKV RNA standard, the limit of detection was 20 copies/test. For the simulated ZIKV clinical samples, the limit of detection was 0.02 pfu/test, which was one order of magnitude higher than RT-PCR and similar to real-time PCR. The detection limit of simulated ZIKV specimens prepared using a protease quick processing method was consistent with that of samples prepared using commercial nucleic acid extraction kits, indicating that our ZIKV detection method could be used in point-of-care testing. The RT-LAMP assay had excellent sensitivity and specificity for detecting ZIKV and can be deployed together with a rapid specimen processing method, offering the possibility for ZIKV diagnosis outside of the laboratory. Copyright © 2016 Elsevier B.V. All rights reserved.

O-5S quantitative real-time PCR: a new diagnostic tool for laboratory confirmation of human onchocerciasis.

PubMed

Mekonnen, Solomon A; Beissner, Marcus; Saar, Malkin; Ali, Solomon; Zeynudin, Ahmed; Tesfaye, Kassahun; Adbaru, Mulatu G; Battke, Florian; Poppert, Sven; Hoelscher, Michael; Löscher, Thomas; Bretzel, Gisela; Herbinger, Karl-Heinz

2017-10-02

Onchocerciasis is a parasitic disease caused by the filarial nematode Onchocerca volvulus. In endemic areas, the diagnosis is commonly confirmed by microscopic examination of skin snip samples, though this technique is considered to have low sensitivity. The available melting-curve based quantitative real-time PCR (qPCR) using degenerated primers targeting the O-150 repeat of O. volvulus was considered insufficient for confirming the individual diagnosis, especially in elimination studies. This study aimed to improve detection of O. volvulus DNA in clinical samples through the development of a highly sensitive qPCR assay. A novel hydrolysis probe based qPCR assay was designed targeting the specific sequence of the O. volvulus O-5S rRNA gene. A total of 200 clinically suspected onchocerciasis cases were included from Goma district in South-west Ethiopia, from October 2012 through May 2013. Skin snip samples were collected and subjected to microscopy, O-150 qPCR, and the novel O-5S qPCR. Among the 200 individuals, 133 patients tested positive (positivity rate of 66.5%) and 67 negative by O-5S qPCR, 74 tested positive by microscopy (37.0%) and 78 tested positive by O-150 qPCR (39.0%). Among the 133 O-5S qPCR positive individuals, microscopy and O-150 qPCR detected 55.6 and 59.4% patients, respectively, implying a higher sensitivity of O-5S qPCR than microscopy and O-150 qPCR. None of the 67 individuals who tested negative by O-5S qPCR tested positive by microscopy or O-150 qPCR, implying 100% specificity of the newly designed O-5S qPCR assay. The novel O-5S qPCR assay is more sensitive than both microscopic examination and the existing O-150 qPCR for the detection of O. volvulus from skin snip samples. The newly designed assay is an important step towards appropriate individual diagnosis and control of onchocerciasis.

Simultaneous detection of bovine and porcine DNA in pharmaceutical gelatin capsules by duplex PCR assay for Halal authentication.

PubMed

Nikzad, Jafar; Shahhosseini, Soraya; Tabarzad, Maryam; Nafissi-Varcheh, Nastaran; Torshabi, Maryam

2017-02-14

In the pharmaceutical industry, hard- and soft-shelled capsules are typically made from gelatin, commonly derived from bovine and porcine sources. To ensure that pharmaceutical products comply with halal regulations in Muslim countries (no porcine products allowed), development of a valid, reliable, quick, and most importantly, cost-effective tests are of utmost importance. We developed a species-specific duplex polymerase chain reaction (PCR) assay targeting 149 bp porcine and 271 bp bovine mitochondrial DNA (mtDNA) to simultaneously detect both porcine and bovine DNA (in one reaction at the same time) in gelatin. Some additional simplex PCR tests (targeting 126 bp bovine and 212 bp porcine mtDNA) and real-time PCR using a commercially available kit (for identification of porcine DNA) were used to verify the selectivity and sensitivity of our duplex PCR. After optimization of DNA extraction and PCR methods, hard/soft pharmaceutical gelatin capsules (containing drug) were tested for the presence of porcine and/or bovine DNA. Duplex PCR detected the presence of as little as 0.1% porcine DNA, which was more accurate than the commercially available kit. Of all gelatin capsules tested (n = 24), 50% contained porcine DNA (pure porcine gelatin alone or in combination with bovine gelatin). Duplex PCR presents an easy-to-follow, quick, low-cost and reliable method to simultaneously detect porcine and bovine DNAs (>100 bp) in minute amounts in highly processed gelatin-containing pharmaceutical products (with a 0.1% sensitivity for porcine DNA) which may be used for halal authentication. Simultaneous detection of porcine and bovine DNA in gelatin capsules by duplex PCR.

FlindersTechnology Associates (FTA) filter paper-based DNA extraction with polymerase chain reaction (PCR) for detection of Pneumocystis jirovecii from respiratory specimens of immunocompromised patients.

PubMed

Nuchprayoon, Surang; Saksirisampant, Wilai; Jaijakul, Siraya; Nuchprayoon, Issarang

2007-01-01

We evaluated the diagnostic value of Flinders Technology Associates (FTA) filter paper together with polymerase chain reaction (PCR) for detection of Pneumocystis jirovecii (carinii) from induced sputum (IS) and bronchoalveolar lavage fluid (BALF) samples. The study involved 162 patients with clinical diagnosis of pneumocystis pneumonia (PcP) of human immunodeficiency virus/acquired immune deficiency syndrome (HIV/AIDS) patients and other immunocompromised patients. P. jirovecii cysts or trophozoites were detected in IS and BALF by cytological method. The mitochondrial 5S ribosomal ribonucleic acid (rRNA) gene of P. jirovecii was amplified from these samples by using FTA filters together with a one-step PCR method (FTA-PCR). With the FTA-PCR method, the sensitivity and specificity of the test compared to microscopic examination were 67% and 90% for IS, while they were 67% and 91% for BALF, respectively. The sensitivity and specificity of the FTA-PCR test was also comparable to PCR with the conventional deoxyribonucleic acid (DNA) extraction method. We concluded that FTA-PCR is useful to detect P. jirovecii in noninvasive IS.

Nested-PCR real time as alternative molecular tool for detection of Borrelia burgdorferi compared to the classical serological diagnosis of the blood.

PubMed

Sroka-Oleksiak, Agnieszka; Ufir, Krzysztof; Salamon, Dominika; Bulanda, Malgorzata; Gosiewski, Tomasz

Lyme disease, caused by Borrelia burgdorferi, is a multisystem disease that often makes difficulties to recognize caused by their genetic heterogenity. Currently, the gold standard for the detection of Lyme disease (LD) is serologic diagnostics based mainly on tests: ELISA and Western blot (WB). These methods, however, are subject to consider- able defect, especially in the initial phase of infection due to the occurrence of so-called serological window period and low specificity. For this reason, they might be replaced by molecular methods, for example polymerase chain reaction (PCR), which should be more sensitivity and specificity. In the present study we attempt to optimize the PCR reaction conditions and enhance existing test sensitivity by applying the equivalent of real time PCR - nested PCR for detection B. burgdorferi DNA in the patient's blood. The study involved 94 blood samples of patients with suspected LD. From each sample, 1.5 ml of blood was used for the isolation of bacterial DNA and PCR real time am- plification and its equivalent, in nested version. The remaining part earmarked for serologi- cal testing. Optimization of the reaction conditions made experimentally, using gradient of the temperature and gradient of the magnesium ions concentration for reaction real time in nested-PCR and PCR version. The results show that the nested-PCR real time, has a much higher sensitivity 45 (47.8%) of positive results for the detection of B. burgdorferi compared to the single- variety, without a preceding pre-amplification 2 (2.1%). Serological methods allowed the detection of infection in 41 (43.6%) samples. These results support of the nested PCR method as a better molecular tool for the detection of B. burgdorferi infection than classical PCR real time reaction. The nested-PCR real time method may be considered as a complement to ELISA and WB mainly in the early stages of infection, when in the blood circulating B. burgdorferi cells. By contrast, the results of serological and molecular tests should always be carried out tak- ing into account the patient's clinical status.

Quantitative PCR detection of Batrachochytrium dendrobatidis DNA from sediments and water

USGS Publications Warehouse

Kirshtein, Julie D.; Anderson, Chauncey W.; Wood, J.S.; Longcore, Joyce E.; Voytek, Mary A.

2007-01-01

The fungal pathogen Batrachochytrium dendrobatidis (Bd) causes chytridiomycosis, a disease implicated in amphibian declines on 5 continents. Polymerase chain reaction (PCR) primer sets exist with which amphibians can be tested for this disease, and advances in sampling techniques allow non-invasive testing of animals. We developed filtering and PCR based quantitative methods by modifying existing PCR assays to detect Bd DNA in water and sediments, without the need for testing amphibians; we tested the methods at 4 field sites. The SYBR based assay using Boyle primers (SYBR/Boyle assay) and the Taqman based assay using Wood primers performed similarly with samples generated in the laboratory (Bd spiked filters), but the SYBR/Boyle assay detected Bd DNA in more field samples. We detected Bd DNA in water from 3 of 4 sites tested, including one pond historically negative for chytridiomycosis. Zoospore equivalents in sampled water ranged from 19 to 454 l-1 (nominal detection limit is 10 DNA copies, or about 0.06 zoospore). We did not detect DNA of Bd from sediments collected at any sites. Our filtering and amplification methods provide a new tool to investigate critical aspects of Bd in the environment. ?? Inter-Research 2007.

Systematic review and meta-analysis: rapid diagnostic tests versus placental histology, microscopy and PCR for malaria in pregnant women.

PubMed

Kattenberg, Johanna H; Ochodo, Eleanor A; Boer, Kimberly R; Schallig, Henk Dfh; Mens, Petra F; Leeflang, Mariska Mg

2011-10-28

During pregnancy, malaria infection with Plasmodium falciparum or Plasmodium vivax is related to adverse maternal health and poor birth outcomes. Diagnosis of malaria, during pregnancy, is complicated by the absence or low parasite densities in peripheral blood. Diagnostic methods, other than microscopy, are needed for detection of placental malaria. Therefore, the diagnostic accuracy of rapid diagnostic tests (RDTs), detecting antigen, and molecular techniques (PCR), detecting DNA, for the diagnosis of Plasmodium infections in pregnancy was systematically reviewed. MEDLINE, EMBASE and Web of Science were searched for studies assessing the diagnostic accuracy of RDTs, PCR, microscopy of peripheral and placental blood and placental histology for the detection of malaria infection (all species) in pregnant women. The results of 49 studies were analysed in metandi (Stata), of which the majority described P. falciparum infections. Although both placental and peripheral blood microscopy cannot reliably replace histology as a reference standard for placental P. falciparum infection, many studies compared RDTs and PCR to these tests. The proportion of microscopy positives in placental blood (sensitivity) detected by peripheral blood microscopy, RDTs and PCR are respectively 72% [95% CI 62-80], 81% [95% CI 55-93] and 94% [95% CI 86-98]. The proportion of placental blood microscopy negative women that were negative in peripheral blood microscopy, RDTs and PCR (specificity) are 98% [95% CI 95-99], 94% [95% CI 76-99] and 77% [95% CI 71-82]. Based on the current data, it was not possible to determine if the false positives in RDTs and PCR are caused by sequestered parasites in the placenta that are not detected by placental microscopy. The findings suggest that RDTs and PCR may have good performance characteristics to serve as alternatives for the diagnosis of malaria in pregnancy, besides any other limitations and practical considerations concerning the use of these tests. Nevertheless, more studies with placental histology as reference test are urgently required to reliably determine the accuracy of RDTs and PCR for the diagnosis of placental malaria. P. vivax-infections have been neglected in diagnostic test accuracy studies of malaria in pregnancy.

Systematic review and meta-analysis: rapid diagnostic tests versus placental histology, microscopy and PCR for malaria in pregnant women

PubMed Central

2011-01-01

Background During pregnancy, malaria infection with Plasmodium falciparum or Plasmodium vivax is related to adverse maternal health and poor birth outcomes. Diagnosis of malaria, during pregnancy, is complicated by the absence or low parasite densities in peripheral blood. Diagnostic methods, other than microscopy, are needed for detection of placental malaria. Therefore, the diagnostic accuracy of rapid diagnostic tests (RDTs), detecting antigen, and molecular techniques (PCR), detecting DNA, for the diagnosis of Plasmodium infections in pregnancy was systematically reviewed. Methods MEDLINE, EMBASE and Web of Science were searched for studies assessing the diagnostic accuracy of RDTs, PCR, microscopy of peripheral and placental blood and placental histology for the detection of malaria infection (all species) in pregnant women. Results The results of 49 studies were analysed in metandi (Stata), of which the majority described P. falciparum infections. Although both placental and peripheral blood microscopy cannot reliably replace histology as a reference standard for placental P. falciparum infection, many studies compared RDTs and PCR to these tests. The proportion of microscopy positives in placental blood (sensitivity) detected by peripheral blood microscopy, RDTs and PCR are respectively 72% [95% CI 62-80], 81% [95% CI 55-93] and 94% [95% CI 86-98]. The proportion of placental blood microscopy negative women that were negative in peripheral blood microscopy, RDTs and PCR (specificity) are 98% [95% CI 95-99], 94% [95% CI 76-99] and 77% [95% CI 71-82]. Based on the current data, it was not possible to determine if the false positives in RDTs and PCR are caused by sequestered parasites in the placenta that are not detected by placental microscopy. Conclusion The findings suggest that RDTs and PCR may have good performance characteristics to serve as alternatives for the diagnosis of malaria in pregnancy, besides any other limitations and practical considerations concerning the use of these tests. Nevertheless, more studies with placental histology as reference test are urgently required to reliably determine the accuracy of RDTs and PCR for the diagnosis of placental malaria. P. vivax-infections have been neglected in diagnostic test accuracy studies of malaria in pregnancy. PMID:22035448

Detection of fungal DNA in human body fluids and tissues during a multistate outbreak of fungal meningitis and other infections.

PubMed

Gade, Lalitha; Scheel, Christina M; Pham, Cau D; Lindsley, Mark D; Iqbal, Naureen; Cleveland, Angela Ahlquist; Whitney, Anne M; Lockhart, Shawn R; Brandt, Mary E; Litvintseva, Anastasia P

2013-05-01

Exserohilum rostratum was the major cause of an outbreak of fungal infections linked to injections of contaminated methylprednisolone acetate. Because almost 14,000 persons were exposed to product that was possibly contaminated with multiple fungal pathogens, there was unprecedented need for a rapid throughput diagnostic test that could detect both E. rostratum and other unusual agents of fungal infection. Here we report development of a novel PCR test that allowed for rapid and specific detection of fungal DNA in cerebrospinal fluid (CSF), other body fluids and tissues of infected individuals. The test relied on direct purification of free-circulating fungal DNA from fluids and subsequent PCR amplification and sequencing. Using this method, we detected Exserohilum rostratum DNA in 123 samples from 114 case-patients (28% of 413 case-patients for whom 627 samples were available), and Cladosporium DNA in one sample from one case-patient. PCR with novel Exserohilum-specific ITS-2 region primers detected 25 case-patients with samples that were negative using broad-range ITS primers. Compared to fungal culture, this molecular test was more sensitive: of 139 case-patients with an identical specimen tested by culture and PCR, E. rostratum was recovered in culture from 19 (14%), but detected by PCR in 41 (29%), showing a diagnostic sensitivity of 29% for PCR compared to 14% for culture in this patient group. The ability to rapidly confirm the etiologic role of E. rostratum in these infections provided an important contribution in the public health response to this outbreak.

Diagnosis of toxoplasmosis after allogeneic stem cell transplantation: results of DNA detection and serological techniques.

PubMed

Fricker-Hidalgo, Hélène; Bulabois, Claude-Eric; Brenier-Pinchart, Marie-Pierre; Hamidfar, Rebecca; Garban, Frédéric; Brion, Jean-Paul; Timsit, Jean-François; Cahn, Jean-Yves; Pelloux, Hervé

2009-01-15

The biological diagnosis of toxoplasmosis after allogeneic hematopoietic stem cell transplantation (HSCT) is based on the detection of Toxoplasma gondii DNA in blood specimens or other samples. Serological testing is used mainly to define the immunity status of the patient before HSCT. The aim of our study was to examine the performance of polymerase chain reaction (PCR) and serological techniques in the diagnosis of toxoplasmosis after HSCT. Seventy patients underwent allogeneic HSCT from September 2004 through September 2006. DNA was detected by PCR, and immunoglobulin G and immunoglobulin M were detected by enzyme-linked immunosorbent assay. The results of immunoglobulin G detection before allogeneic HSCT were positive in 40 (57.1%) of the patients and negative in 30 (42.9%). After HSCT, 57 patients (81.4%) had test results that were negative for immunoglobulin M and had negative results of DNA detection, without toxoplasmosis infection. Four patients (5.7%) had at least 4 samples with positive PCR results and/or test results positive for immunoglobulin M against T. gondii; toxoplasmosis was then confirmed by clinical symptoms. Nine patients (12.9%) with positive PCR results and 1 or 2 samples with test results negative for immunoglobulin M were considered to have asymptomatic T. gondii infection. Reactivation of latent infection was the cause of toxoplasmosis in 3 of the 4 patients, and toxoplasmosis occurred as a primary infection in 1 patient. The detection of specific anti-T. gondii immunoglobulin M was the only biological evidence of toxoplasmosis in 2 patients, and samples were positive for immunoglobulin M before PCR was performed in 1 patient. Thus, after HSCT, all patients were at risk for toxoplasmosis; all patients who receive HSCTs should be followed up with biological testing that combines PCR and serological techniques.

Comparison between nasopharyngeal swab and nasal wash, using culture and PCR, in the detection of potential respiratory pathogens.

PubMed

Gritzfeld, Jenna F; Roberts, Paul; Roche, Lorna; El Batrawy, Sherouk; Gordon, Stephen B

2011-04-13

Nasopharyngeal carriage of potential pathogens is important as it is both the major source of transmission and the prerequisite of invasive disease. New methods for detecting carriage could improve comfort, accuracy and laboratory utility. The aims of this study were to compare the sensitivities of a nasopharyngeal swab (NPS) and a nasal wash (NW) in detecting potential respiratory pathogens in healthy adults using microbiological culture and PCR. Healthy volunteers attended for nasal washing and brushing of the posterior nasopharynx. Conventional and real-time PCR were used to detect pneumococcus and meningococcus. Statistical differences between the two nasal sampling methods were determined using a nonparametric Mann-Whitney U test; differences between culture and PCR methods were determined using the McNemar test.Nasal washing was more comfortable for volunteers than swabbing (n = 24). In detection by culture, the NW was significantly more likely to detect pathogens than the NPS (p < 0.00001). Overall, there was a low carriage rate of pathogens in this sample; no significant difference was seen in the detection of bacteria between culture and PCR methods. Nasal washing and PCR may provide effective alternatives to nasopharyngeal swabbing and classical microbiology, respectively.

Relative Accuracy of Nucleic Acid Amplification Tests and Culture in Detecting Chlamydia in Asymptomatic Men

PubMed Central

Cheng, Hong; Macaluso, Maurizio; Vermund, Sten H.; Hook, Edward W.

2001-01-01

Published estimates of the sensitivity and specificity of PCR and ligase chain reaction (LCR) for detecting Chlamydia trachomatis are potentially biased because of study design limitations (confirmation of test results was limited to subjects who were PCR or LCR positive but culture negative). Relative measures of test accuracy are less prone to bias in incomplete study designs. We estimated the relative sensitivity (RSN) and relative false-positive rate (RFP) for PCR and LCR versus cell culture among 1,138 asymptomatic men and evaluated the potential bias of RSN and RFP estimates. PCR and LCR testing in urine were compared to culture of urethral specimens. Discordant results (PCR or LCR positive, but culture negative) were confirmed by using a sequence including the other DNA amplification test, direct fluorescent antibody testing, and a DNA amplification test to detect chlamydial major outer membrane protein. The RSN estimates for PCR and LCR were 1.45 (95% confidence interval [CI] = 1.3 to 1.7) and 1.49 (95% CI = 1.3 to 1.7), respectively, indicating that both methods are more sensitive than culture. Very few false-positive results were found, indicating that the specificity levels of PCR, LCR, and culture are high. The potential bias in RSN and RFP estimates were <5 and <20%, respectively. The estimation of bias is based on the most likely and probably conservative parameter settings. If the sensitivity of culture is between 60 and 65%, then the true sensitivity of PCR and LCR is between 90 and 97%. Our findings indicate that PCR and LCR are significantly more sensitive than culture, while the three tests have similar specificities. PMID:11682509

Polymerase chain reaction assay for the detection of Helicobacter pylori in gastric biopsy specimens: comparison with culture, rapid urease test, and histopathological tests.

PubMed Central

Fabre, R; Sobhani, I; Laurent-Puig, P; Hedef, N; Yazigi, N; Vissuzaine, C; Rodde, I; Potet, F; Mignon, M; Etienne, J P

1994-01-01

Ulcer recurrence is probably related to residual Helicobacter pylori (H pylori). Histological examination and culture are considered to be the most specific tests. CLO test is a rapid but less specific test, which is usually used as an alternative test to culture. The aim of this study was to investigate the efficiency of a simplified polymerase chain reaction (PCR) assay as a procedure for the diagnosis of gastric H pylori infection of patients. Biopsy specimens were obtained from antral mucosa of 58 patients at endoscopy and submitted to four tests for detection of H pylori. The bacteria were found in 53%, 43%, 48%, and 50% of patients according to the results of PCR, CLO test, culture, and histological examination. Twenty three patients had both negative histology and negative culture and PCR was negative in all of these. Thirteen patients were not classified because only histology or culture was positive and 10 of these had a positive PCR test. When the diagnosis of H pylori was established by agreement with both histology and culture or three positive tests out of four, 29 patients were H pylori positive (28 having had three positive tests and one displaying positive histology and culture), and 26 were negative, and three undetermined. PCR proved the most sensitive and specific test. These results suggest the simplified PCR assay may be a valuable test for the detection of H pylori. Images p906-a PMID:8063217

Multiplex PCR for Rapid Detection of Genes Encoding Class A Carbapenemases

PubMed Central

Hong, Sang Sook; Kim, Kyeongmi; Huh, Ji Young; Jung, Bochan; Kang, Myung Seo

2012-01-01

In recent years, there have been increasing reports of KPC-producing Klebsiella pneumoniae in Korea. The modified Hodge test can be used as a phenotypic screening test for class A carbapenamase (CAC)-producing clinical isolates; however, it does not distinguish between carbapenemase types. The confirmation of type of CAC is important to ensure optimal therapy and to prevent transmission. This study applied a novel multiplex PCR assay to detect and differentiate CAC genes in a single reaction. Four primer pairs were designed to amplify fragments encoding 4 CAC families (SME, IMI/NMC-A, KPC, and GES). The multiplex PCR detected all genes tested for 4 CAC families that could be differentiated by fragment size according to gene type. This multiplex PCR offers a simple and useful approach for detecting and distinguishing CAC genes in carbapenem-resistant strains that are metallo-β-lactamase nonproducers. PMID:22950072

Multiplex PCR for rapid detection of genes encoding class A carbapenemases.

PubMed

Hong, Sang Sook; Kim, Kyeongmi; Huh, Ji Young; Jung, Bochan; Kang, Myung Seo; Hong, Seong Geun

2012-09-01

In recent years, there have been increasing reports of KPC-producing Klebsiella pneumoniae in Korea. The modified Hodge test can be used as a phenotypic screening test for class A carbapenamase (CAC)-producing clinical isolates; however, it does not distinguish between carbapenemase types. The confirmation of type of CAC is important to ensure optimal therapy and to prevent transmission. This study applied a novel multiplex PCR assay to detect and differentiate CAC genes in a single reaction. Four primer pairs were designed to amplify fragments encoding 4 CAC families (SME, IMI/NMC-A, KPC, and GES). The multiplex PCR detected all genes tested for 4 CAC families that could be differentiated by fragment size according to gene type. This multiplex PCR offers a simple and useful approach for detecting and distinguishing CAC genes in carbapenem-resistant strains that are metallo-β-lactamase nonproducers.

Rapid and sensitive detection of Yersinia pestis using amplification of plague diagnostic bacteriophages monitored by real-time PCR.

PubMed

Sergueev, Kirill V; He, Yunxiu; Borschel, Richard H; Nikolich, Mikeljon P; Filippov, Andrey A

2010-06-28

Yersinia pestis, the agent of plague, has caused many millions of human deaths and still poses a serious threat to global public health. Timely and reliable detection of such a dangerous pathogen is of critical importance. Lysis by specific bacteriophages remains an essential method of Y. pestis detection and plague diagnostics. The objective of this work was to develop an alternative to conventional phage lysis tests--a rapid and highly sensitive method of indirect detection of live Y. pestis cells based on quantitative real-time PCR (qPCR) monitoring of amplification of reporter Y. pestis-specific bacteriophages. Plague diagnostic phages phiA1122 and L-413C were shown to be highly effective diagnostic tools for the detection and identification of Y. pestis by using qPCR with primers specific for phage DNA. The template DNA extraction step that usually precedes qPCR was omitted. phiA1122-specific qPCR enabled the detection of an initial bacterial concentration of 10(3) CFU/ml (equivalent to as few as one Y. pestis cell per 1-microl sample) in four hours. L-413C-mediated detection of Y. pestis was less sensitive (up to 100 bacteria per sample) but more specific, and thus we propose parallel qPCR for the two phages as a rapid and reliable method of Y. pestis identification. Importantly, phiA1122 propagated in simulated clinical blood specimens containing EDTA and its titer rise was detected by both a standard plating test and qPCR. Thus, we developed a novel assay for detection and identification of Y. pestis using amplification of specific phages monitored by qPCR. The method is simple, rapid, highly sensitive, and specific and allows the detection of only live bacteria.

PCR detection and quantitation of predominant anaerobic bacteria in human and animal fecal samples.

PubMed Central

Wang, R F; Cao, W W; Cerniglia, C E

1996-01-01

PCR procedures based on 16S rRNA gene sequences specific for 12 anaerobic bacteria that predominate in the human intestinal tract were developed and used for quantitative detection of these species in human (adult and baby) feces and animal (rat, mouse, cat, dog, monkey, and rabbit) feces. Fusobacterium prausnitzii, Peptostreptococcus productus, and Clostridium clostridiiforme had high PCR titers (the maximum dilutions for positive PCR results ranged from 10(-3) to 10(-8)) in all of the human and animal fecal samples tested. Bacteroides thetaiotaomicron, Bacteroides vulgatus, and Eubacterium limosum also showed higher PCR titers (10(-2) to 10(-6)) in adult human feces. The other bacteria tested, including Escherichia coli, Bifidobacterium adolescentis, Bifidobacterium longum, Lactobacillus acidophilus, Eubacterium biforme, and Bacteroides distasonis, were either at low PCR titers (less than 10(-2)) or not detected by PCR. The reported PCR procedure including the fecal sample preparation method is simplified and rapid and eliminates the DNA isolation steps. PMID:8919784

Methods for Integrated Air Sampling and DNA Analysis for Detection of Airborne Fungal Spores

PubMed Central

Williams, Roger H.; Ward, Elaine; McCartney, H. Alastair

2001-01-01

Integrated air sampling and PCR-based methods for detecting airborne fungal spores, using Penicillium roqueforti as a model fungus, are described. P. roqueforti spores were collected directly into Eppendorf tubes using a miniature cyclone-type air sampler. They were then suspended in 0.1% Nonidet P-40, and counted using microscopy. Serial dilutions of the spores were made. Three methods were used to produce DNA for PCR tests: adding untreated spores to PCRs, disrupting spores (fracturing of spore walls to release the contents) using Ballotini beads, and disrupting spores followed by DNA purification. Three P. roqueforti-specific assays were tested: single-step PCR, nested PCR, and PCR followed by Southern blotting and probing. Disrupting the spores was found to be essential for achieving maximum sensitivity of the assay. Adding untreated spores to the PCR did allow the detection of P. roqueforti, but this was never achieved when fewer than 1,000 spores were added to the PCR. By disrupting the spores, with or without subsequent DNA purification, it was possible to detect DNA from a single spore. When known quantities of P. roqueforti spores were added to air samples consisting of high concentrations of unidentified fungal spores, pollen, and dust, detection sensitivity was reduced. P. roqueforti DNA could not be detected using untreated or disrupted spore suspensions added to the PCRs. However, using purified DNA, it was possible to detect 10 P. roqueforti spores in a background of 4,500 other spores. For all DNA extraction methods, nested PCR was more sensitive than single-step PCR or PCR followed by Southern blotting. PMID:11375150

Comparison of FilmArray and Quantitative Real-Time Reverse Transcriptase PCR for Detection of Zaire Ebolavirus from Contrived and Clinical Specimens

PubMed Central

Southern, Timothy R.; Racsa, Lori D.; Albariño, César G.; Fey, Paul D.; Hinrichs, Steven H.; Murphy, Caitlin N.; Herrera, Vicki L.; Sambol, Anthony R.; Hill, Charles E.; Ryan, Emily L.; Kraft, Colleen S.; Campbell, Shelley; Sealy, Tara K.; Schuh, Amy; Ritchie, James C.; Lyon, G. Marshall; Mehta, Aneesh K.; Varkey, Jay B.; Ribner, Bruce S.; Brantly, Kent P.; Ströher, Ute; Iwen, Peter C.

2015-01-01

Rapid, reliable, and easy-to-use diagnostic assays for detection of Zaire ebolavirus (ZEBOV) are urgently needed. The goal of this study was to examine the agreement among emergency use authorization (EUA) tests for the detection of ZEBOV nucleic acids, including the BioFire FilmArray BioThreat (BT) panel, the FilmArray BT-E panel, and the NP2 and VP40 quantitative real-time reverse transcriptase (qRT) PCR assays from the Centers for Disease Control and Prevention (CDC). Specimens used in this study included whole blood spiked with inactivated ZEBOV at known titers and whole-blood, plasma, and urine clinical specimens collected from persons diagnosed with Ebola virus disease (EVD). The agreement for FilmArray and qRT-PCR results using contrived whole-blood specimens was 100% (6/6 specimens) for each ZEBOV dilution from 4 × 107 to 4 × 102 50% tissue culture infective dose (TCID50)/ml, as well as the no-virus negative-control sample. The limit of detection for FilmArray and qRT-PCR assays with inactivated ZEBOV, based on duplicate positive results, was determined to be 4 × 102 TCID50/ml. Rates of agreement between FilmArray and qRT-PCR results for clinical specimens from patients with EVD were 85% (23/27 specimens) for whole-blood specimens, 90% (18/20 specimens) for whole-blood specimens tested by FilmArray testing and matched plasma specimens tested by qRT-PCR testing, and 85% (11/13 specimens) for urine specimens. Among 60 specimens, eight discordant results were noted, with ZEBOV nucleic acids being detected only by FilmArray testing in four specimens and only by qRT-PCR testing in the remaining four specimens. These findings demonstrate that the rapid and easy-to-use FilmArray panels are effective tests for evaluating patients with EVD. PMID:26157148

The agony of choice in dermatophyte diagnostics-performance of different molecular tests and culture in the detection of Trichophyton rubrum and Trichophyton interdigitale.

PubMed

Kupsch, C; Ohst, T; Pankewitz, F; Nenoff, P; Uhrlaß, S; Winter, I; Gräser, Y

2016-08-01

Dermatophytosis caused by dermatophytes of the genera Trichophyton and Microsporum belong to the most frequent mycoses worldwide. Molecular detection methods proved to be highly sensitive and enable rapid and accurate detection of dermatophyte species from clinical specimens. For the first time, we compare the performance of different molecular methods with each other and with conventional diagnostics in the detection of dermatophytoses caused by Trichophyton rubrum and Trichophyton interdigitale in clinical specimens (nail, skin and hair). The compared molecular methods comprise two already published PCR-ELISAs, a published quantitative RT-PCR as well as a newly developed PCR-ELISA targeting the internal transcribed spacer region. We investigated the sensitivity of the assays by analysing 375 clinical samples. In 148 specimens (39.5%) a positive result was gained in at least one of the four molecular tests or by culture, but the number of detected agents differed significantly between some of the assays. The most sensitive assay, a PCR-ELISA targeting a microsatellite region, detected 81 T. rubrum infections followed by an internal transcribed spacer PCR-ELISA (60), quantitative RT-PCR (52) and a topoisomerase II PCR-ELISA (51), whereas cultivation resulted in T. rubrum identification in 37 samples. The pros and cons of all four tests in routine diagnostics are discussed. Copyright © 2016 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Roche/BIOTECON Diagnostics LightCycler foodproof L. monocytogenes detection kit in combination with ShortPrep foodproof II Kit. Performance-Tested Method 070401.

PubMed

Junge, Benjamin; Berghof-Jäger, Kornelia

2006-01-01

A method was developed for the detection of L. monocytogenes in food based on real-time polymerase chain reaction (PCR). This advanced PCR method was designed to reduce the time needed to achieve results from PCR reactions and to enable the user to monitor the amplification of the PCR product simultaneously, in real-time. After DNA isolation using the Roche/BIOTECON Diagnostics ShortPrep foodproof II Kit (formerly called Listeria ShortPrep Kit) designed for the rapid preparation of L. monocytogenes DNA for direct use in PCR, the real-time detection of L. monocytogenes DNA is performed by using the Roche/BIOTECON Diagnostics LightCycler foodproof L. monocytogenes Detection Kit. This kit provides primers and hybridization probes for sequence-specific detection, convenient premixed reagents, and different controls for reliable interpretation of results. For repeatability studies, 20 different foods, covering the 15 food groups recommended from the AOAC Research Institute (AOAC RI) for L. monocytogenes detection were analyzed: raw meats, fresh produce/vegetables, processed meats, seafood, egg and egg products, dairy (cultured/noncultured), spices, dry foods, fruit/juices, uncooked pasta, nuts, confectionery, pet food, food dyes and colorings, and miscellaneous. From each food 20, samples were inoculated with a low level (1-10 colony-forming units (CFU)/25 g) and 20 samples with a high level (10-50 CFU/25 g) of L. monocytogenes. Additionally, 5 uninoculated samples were prepared from each food. The food samples were examined with the test kits and in correlation with the cultural methods according to U.S. Food and Drug Administration (FDA) Bacteriological Analytical Manual (BAM) or U.S. Department of Agriculture (USDA)/Food Safety and Inspection Service (FSIS) Microbiology Laboratory Guidebook. After 48 h of incubation, the PCR method in all cases showed equal or better results than the reference cultural FDA/BAM or USDA/FSIS methods. Fifteen out of 20 tested food types gave exactly the same amount of positive samples for both methods in both inoculation levels. For 5 out of 20 foodstuffs, the PCR method resulted in more positives than the reference method after 48 h of incubation. Following AOAC RI definition, these were false positives because they were not confirmed by the reference method (false-positive rate for low inoculated foodstuffs: 5.4%; for high inoculated foodstuffs: 7.1%). Without calculating these unconfirmed positives, the PCR method showed equal sensitivity results compared to the alternative method. With the unconfirmed PCR-positives included into the calculations, the alternative PCR method showed a higher sensitivity than the microbiological methods (low inoculation level: 100 vs 98.0%; sensitivity rate: 1; high inoculation level: 99.7 vs 97.7%; sensitivity rate, 1). All in-house and independently tested uninoculated food samples were negative for L. monocytogenes. The ruggedness testing of both ShortPrep foodproof II Kit and Roche/BIOTECON LightCycler foodproof L. monocytogenes Detection Kit showed no noteworthy influences to any variation of the parameters component concentration, apparatus comparison, tester comparison, and sample volumes. In total, 102 L. monocytogenes isolates (cultures and pure DNA) were tested and detected for the inclusivity study, including all isolates claimed by the AOAC RI. The exclusivity study included 60 non-L. monocytogenes bacteria. None of the tested isolates gave a false-positive result; specificity was 100%. Three different lots were tested in the lot-to-lot study. All 3 lots gave equal results. The stability study was subdivided into 3 parts: long-term study, stress test, and freeze-defrost test. Three lots were tested in 4 time intervals within a period of 13 months. They all gave comparable results for all test intervals. For the stress test, LightCycler L. monocytogenes detection mixes were stored at different temperatures and tested at different time points during 1 month. Stable results were produced at all storage temperatures. The freeze-defrost analysis showed no noteworthy aggravation of test results. The independent validation study examined by Campden and Chorleywood Food Research Association Group (CCFRA) demonstrated again that the LightCycler L. monocytogenes detection system shows a comparable sensitivity to reference methods. With both the LightCycler PCR and BAM methods, 19 out of 20 inoculated food samples were detected. The 24 h PCR results generated by the LightCycler system corresponded directly with the FDA/BAM culture results. However, the 48 h PCR results did not relate exactly to the FDA/BAM results, as one sample found to be positive by the 48 h PCR could not be culturally confirmed and another sample which was negative by the 48 h PCR was culturally positive.

Review: Diagnostic accuracy of PCR-based detection tests for Helicobacter Pylori in stool samples.

PubMed

Khadangi, Fatemeh; Yassi, Maryam; Kerachian, Mohammad Amin

2017-12-01

Although different methods have been established to detect Helicobacter pylori (H. pylori) infection, identifying infected patients is an ongoing challenge. The aim of this meta-analysis was to provide pooled diagnostic accuracy measures for stool PCR test in the diagnosis of H. pylori infection. In this study, a systematic review and meta-analysis were carried out on various sources, including MEDLINE, Web of Sciences, and the Cochrane Library from April 1, 1999, to May 1, 2016. This meta-analysis adheres to the guidelines provided by the Preferred Reporting Items for Systematic Reviews and Meta-Analyses report (PRISMA Statement). The clinical value of DNA stool PCR test was based on the pooled false positive, false negative, true positive, and true negative of different genes. Twenty-six of 328 studies identified met the eligibility criteria. Stool PCR test had a performance of 71% (95% CI: 68-73) sensitivity, 96% (95% CI: 94-97) specificity, and 65.6 (95% CI: 30.2-142.5) diagnostic odds ratio (DOR) in diagnosis of H. pylori. The DOR of genes which showed the highest performance of stool PCR tests was as follows: 23S rRNA 152.5 (95% CI: 55.5-418.9), 16S rRNA 67.9 (95%CI: 6.4-714.3), and glmM 68.1 (95%CI: 20.1-231.7). The sensitivity and specificity of stool PCR test are relatively in the same spectrum of other diagnostic methods for the detection of H. pylori infection. In descending order of significance, the most diagnostic candidate genes using PCR detection were 23S rRNA, 16S rRNA, and glmM. PCR for 23S rRNA gene which has the highest performance could be applicable to detect H. pylori infection. © 2017 John Wiley & Sons Ltd.

Development of a highly sensitive real-time nested RT-PCR assay in a single closed tube for detection of enterovirus 71 in hand, foot, and mouth disease.

PubMed

Niu, Peihua; Qi, Shunxiang; Yu, Benzhang; Zhang, Chen; Wang, Ji; Li, Qi; Ma, Xuejun

2016-11-01

Enterovirus 71 (EV71) is one of the major causative agents of outbreaks of hand, foot, and mouth disease (HFMD). A commercial TaqMan probe-based real-time PCR assay has been widely used for the differential detection of EV71 despite its relatively high cost and failure to detect samples with a low viral load (Ct value > 35). In this study, a highly sensitive real-time nested RT-PCR (RTN RT-PCR) assay in a single closed tube for detection of EV71 in HFMD was developed. The sensitivity and specificity of this assay were evaluated using a reference EV71 stock and a panel of controls consisting of coxsackievirus A16 (CVA16) and common respiratory viruses, respectively. The clinical performance of this assay was evaluated and compared with those of a commercial TaqMan probe-based real-time PCR (qRT-PCR) assay and a traditional two-step nested RT-PCR assay. The limit of detection for the RTN RT-PCR assay was 0.01 TCID50/ml, with a Ct value of 38.3, which was the same as that of the traditional two-step nested RT-PCR assay and approximately tenfold lower than that of the qRT-PCR assay. When testing the reference strain EV71, this assay showed favorable detection reproducibility and no obvious cross-reactivity. The testing results of 100 clinical throat swabs from HFMD-suspected patients revealed that 41 samples were positive for EV71 by both RTN RT-PCR and traditional two-step nested RT-PCR assays, whereas only 29 were EV71 positive by qRT-PCR assay.

Rapid detection of fungal keratitis with DNA-stabilizing FTA filter paper.

PubMed

Menassa, Nardine; Bosshard, Philipp P; Kaufmann, Claude; Grimm, Christian; Auffarth, Gerd U; Thiel, Michael A

2010-04-01

Purpose. Polymerase chain reaction (PCR) is increasingly important for the rapid detection of fungal keratitis. However, techniques of specimen collection and DNA extraction before PCR may interfere with test sensitivity. The purpose of this study was to investigate the use of DNA-stabilizing FTA filter paper (Indicating FTA filter paper; Whatman International, Ltd., Maidstone, UK) for specimen collection without DNA extraction in a single-step, nonnested PCR for fungal keratitis. Methods. Specimens were collected from ocular surfaces with FTA filter discs, which automatically lyse collected cells and stabilize nucleic acids. Filter discs were directly used in single-step PCR reactions to detect fungal DNA. Test sensitivity was evaluated with serial dilutions of Candida albicans, Fusarium oxysporum, and Aspergillus fumigatus cultures. Test specificity was analyzed by comparing 196 and 155 healthy individuals from Switzerland and Egypt, respectively, with 15 patients with a diagnosis of microbial keratitis. Results. PCR with filter discs detected 3 C. albicans, 25 F. oxysporum, and 125 A. fumigatus organisms. In healthy volunteers, fungal PCR was positive in 1.0% and 8.4% of eyes from Switzerland and Egypt, respectively. Fungal PCR remained negative in 10 cases of culture-proven bacterial keratitis, became positive in 4 cases of fungal keratitis, but missed 1 case of culture-proven A. fumigatus keratitis. Conclusions. FTA filter paper for specimen collection together with direct PCR is a promising method of detecting fungal keratitis. The analytical sensitivity is high without the need for a semi-nested or nested second PCR, the clinical specificity is 91.7% to 99.0%, and the method is rapid and inexpensive.

Direct Fluorescence Detection of Allele-Specific PCR Products Using Novel Energy-Transfer Labeled Primers.

PubMed

Winn-Deen

1998-12-01

Background: Currently analysis of point mutations can be done by allele-specific polymerase chain reaction (PCR) followed by gel analysis or by gene-specific PCR followed by hybridization with an allele-specific probe. Both of these mutation detection methods require post-PCR laboratory time and run the risk of contaminating subsequent experiments with the PCR product liberated during the detection step. The author has combined the PCR amplification and detection steps into a single procedure suitable for closed-tube analysis. Methods and Results: Allele-specific PCR primers were designed as Sunrise energy-transfer primers and contained a 3' terminal mismatch to distinguish between normal and mutant DNA. Cloned normal (W64) and mutant (R64) templates of the beta3-adrenergic receptor gene were tested to verify amplification specificity and yield. A no-target negative control was also run with each reaction. After PCR, each reaction was tested for fluorescence yield by measuring fluorescence on a spectrofluorimeter or fluorescent microtitreplate reader. The cloned controls and 24 patient samples were tested for the W64R mutation by two methods. The direct fluorescence results with the Sunrise allele-specific PCR method gave comparable genotypes to those obtained with the PCR/ restriction digest/gel electrophoresis control method. No PCR artifacts were observed in the negative controls or in the PCR reactions run with the mismatched target. Conclusions: The results of this pilot study indicate good PCR product and fluorescence yield from allele-specific energy-transfer labeled primers, and the capability of distinguishing between normal and mutant alleles based on fluorescence alone, without the need for restriction digestion, gel electrophoresis, or hybridization with an allele-specific probe.

Development of an updated PCR assay for detection of African swine fever virus.

PubMed

Luo, Yuzi; Atim, Stella A; Shao, Lina; Ayebazibwe, Chrisostom; Sun, Yuan; Liu, Yan; Ji, Shengwei; Meng, Xing-Yu; Li, Su; Li, Yongfeng; Masembe, Charles; Ståhl, Karl; Widén, Frederik; Liu, Lihong; Qiu, Hua-Ji

2017-01-01

Due to the current unavailability of vaccines or treatments for African swine fever (ASF), which is caused by African swine fever virus (ASFV), rapid and reliable detection of the virus is essential for timely implementation of emergency control measures and differentiation of ASF from other swine diseases with similar clinical presentations. Here, an improved PCR assay was developed and evaluated for sensitive and universal detection of ASFV. Primers specific for ASFV were designed based on the highly conserved region of the vp72 gene sequences of all ASFV strains available in GenBank, and the PCR assay was established and compared with two OIE-validated PCR tests. The analytic detection limit of the PCR assay was 60 DNA copies per reaction. No amplification signal was observed for several other porcine viruses. The novel PCR assay was more sensitive than two OIE-validated PCR assays when testing 14 strains of ASFV representing four genotypes (I, V, VIII and IX) from diverse geographical areas. A total of 62 clinical swine blood samples collected from Uganda were examined by the novel PCR, giving a high agreement (59/62) with a superior sensitive universal probe library-based real-time PCR. Eight out of 62 samples tested positive, and three samples with higher Ct values (39.15, 38.39 and 37.41) in the real-time PCR were negative for ASFV in the novel PCR. In contrast, one (with a Ct value of 29.75 by the real-time PCR) and two (with Ct values of 29.75 and 33.12) ASFV-positive samples were not identified by the two OIE-validated PCR assays, respectively. Taken together, these data show that the novel PCR assay is specific, sensitive, and applicable for molecular diagnosis and surveillance of ASF.

Duration of Group A Streptococcus PCR positivity following antibiotic treatment of pharyngitis.

PubMed

Homme, Jason H; Greenwood, Corryn S; Cronk, Lisa B; Nyre, Lisa M; Uhl, James R; Weaver, Amy L; Patel, Robin

2018-02-01

Polymerase chain reaction (PCR) has high sensitivity and specificity for detection of group A streptococcus (GAS) in throat swabs and is routinely used for GAS pharyngitis diagnosis at our institution. Herein we defined the natural history of throat swab GAS PCR and culture positivity during and following treatment of GAS pharyngitis. Fifty children with a PCR positive GAS throat swab were recruited for participation. Four additional throat swabs were collected over 2 weeks following the initial positive PCR result (during and following a standard course of antibiotic therapy) and tested for GAS using rapid real-time PCR and culture. After the initial positive swab, 45% had a positive PCR 2-4 days, 20% 5-7 days, 18% 8-10 days, 25% 11-13days, and 20% 14-18days later. The median time to a negative PCR was 4 days with the nadir in positive PCR results approximating the end of a typical 10-day treatment interval. Seven subjects remained persistently PCR positive. Culture results remained positive at a stable rate for each time interval, ranging from 5-10%. If a patient presents with symptoms of GAS pharyngitis after previous positive GAS PCR testing and treatment with appropriate antibiotics, it is reasonable to use PCR testing for GAS pharyngitis testing beginning one week after initial testing. Further studies are warranted to determine if this time frame can be applied to PCR testing used to detect other infections. Copyright © 2017 Elsevier Inc. All rights reserved.

Development of real-time RT-PCR for the detection of low concentrations of Rift Valley fever virus.

PubMed

Maquart, Marianne; Temmam, Sarah; Héraud, Jean-Michel; Leparc-Goffart, Isabelle; Cêtre-Sossah, Catherine; Dellagi, Koussay; Cardinale, Eric; Pascalis, Hervé

2014-01-01

In recent years, Madagascar and the Comoros archipelago have been affected by epidemics of Rift Valley fever (RVF), however detection of Rift Valley fever virus (RVFV) in zebu, sheep and goats during the post epidemic periods was frequently unsuccessful. Thus, a highly sensitive real-time RT-PCR assay was developed for the detection of RVFV at low viral loads. A new RVF SYBR Green RT-PCR targeting the M segment was tested on serum from different RVF seronegative ruminant species collected from May 2010 to August 2011 in Madagascar and the Comoros archipelago and compared with a RVF specific quantitative real time RT-PCR technique, which is considered as the reference technique. The specificity was tested on a wide range of arboviruses or other viruses giving RVF similar clinical signs. A total of 38 out of 2756 serum samples tested positive with the new RT-PCR, whereas the reference technique only detected 5 out of the 2756. The described RT-PCR is an efficient diagnostic tool for the investigation of enzootic circulation of the RVF virus. It allows the detection of low viral RNA loads adapted for the investigations of reservoirs or specific epidemiological situations such as inter-epizootic periods. Copyright © 2013 Elsevier B.V. All rights reserved.

Detecting Malaria Hotspots: A Comparison of Rapid Diagnostic Test, Microscopy, and Polymerase Chain Reaction.

PubMed

Mogeni, Polycarp; Williams, Thomas N; Omedo, Irene; Kimani, Domtila; Ngoi, Joyce M; Mwacharo, Jedida; Morter, Richard; Nyundo, Christopher; Wambua, Juliana; Nyangweso, George; Kapulu, Melissa; Fegan, Gregory; Bejon, Philip

2017-11-27

Malaria control strategies need to respond to geographical hotspots of transmission. Detection of hotspots depends on the sensitivity of the diagnostic tool used. We conducted cross-sectional surveys in 3 sites within Kilifi County, Kenya, that had variable transmission intensities. Rapid diagnostic test (RDT), microscopy, and polymerase chain reaction (PCR) were used to detect asymptomatic parasitemia, and hotspots were detected using the spatial scan statistic. Eight thousand five hundred eighty-one study participants were surveyed in 3 sites. There were statistically significant malaria hotspots by RDT, microscopy, and PCR for all sites except by microscopy in 1 low transmission site. Pooled data analysis of hotspots by PCR overlapped with hotspots by microscopy at a moderate setting but not at 2 lower transmission settings. However, variations in degree of overlap were noted when data were analyzed by year. Hotspots by RDT were predictive of PCR/microscopy at the moderate setting, but not at the 2 low transmission settings. We observed long-term stability of hotspots by PCR and microscopy but not RDT. Malaria control programs may consider PCR testing to guide asymptomatic malaria hotspot detection once the prevalence of infection falls. © The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America.

Bat white-nose syndrome: a real-time TaqMan polymerase chain reaction test targeting the intergenic spacer region of Geomyces destructanstructans.

USGS Publications Warehouse

Muller, Laura K.; Lorch, Jeffrey M.; Lindner, Daniel L.; O'Connor, Michael; Gargas, Andrea; Blehert, David S.

2013-01-01

The fungus Geomyces destructans is the causative agent of white-nose syndrome (WNS), a disease that has killed millions of North American hibernating bats. We describe a real-time TaqMan PCR test that detects DNA from G. destructans by targeting a portion of the multicopy intergenic spacer region of the rRNA gene complex. The test is highly sensitive, consistently detecting as little as 3.3 fg of genomic DNA from G. destructans. The real-time PCR test specifically amplified genomic DNA from G. destructans but did not amplify target sequence from 54 closely related fungal isolates (including 43 Geomyces spp. isolates) associated with bats. The test was further qualified by analyzing DNA extracted from 91 bat wing skin samples, and PCR results matched histopathology findings. These data indicate the real-time TaqMan PCR method described herein is a sensitive, specific, and rapid test to detect DNA from G. destructans and provides a valuable tool for WNS diagnostics and research.

The Role of PCR in the Diagnosis of Candida Vulvovaginitis-a New Gold Standard?

PubMed

Sobel, J D; Akins, Robert A

2015-06-01

PCR is recognized as a reliable technique for detection of all types of microorganisms. Being highly objective and reproducible also sensitive and specific, PCR is now widely used for sexually transmitted infection (STI) diagnosis. Potential, however, exists for detecting non-pathogens, and not identifying a pathogenic state decreases specificity or clinical significance. PCR Candida tests of vaginal specimens are now widely available and frequently used offering a modest to moderate increase in sensitivity and are likely to replace traditional culture and DNA homology testing. Nevertheless, there remain considerable gaps in our knowledge regarding the usefulness and applications of these expensive tests.

Amplification of Mycoplasma haemofelis DNA by a PCR for point-of-care use.

PubMed

Hawley, Jennifer; Yaaran, Tal; Maurice, Sarah; Lappin, Michael R

2018-01-01

We compared a qualitative in-clinic (IC)-PCR for the detection of Mycoplasma haemofelis DNA with the results of a commercial qualitative laboratory-based, conventional (c)PCR. In order to determine the specificity of both tests, Bartonella spp. samples were included. Forty-three previously tested blood samples with known PCR results for hemoplasmas and Bartonella spp. were selected. The samples were split between 2 laboratories. At the first laboratory, DNA was purified and run on 2 cPCR assays for the detection of hemoplasmas and Bartonella spp. At the second laboratory, DNA was purified using 2 purification protocols and both run in the IC-PCR assay. The cPCR results confirmed that 18 samples were positive for M. haemofelis, 5 for ' Candidatus M. haemominutum', 8 for Bartonella henselae, 2 for Bartonella clarridgeiae, and 10 were negative for both genera. No mixed infections were observed. The IC-PCR assay for the detection of M. haemofelis had a sensitivity of 94.4% and specificity of 96%, when using the same DNA purification method as the first laboratory. Using the second purification method, the sensitivity of the IC-PCR assay was 77.8% and specificity was 96%. Bartonella species were not detected by the IC-PCR M. haemofelis assay. The IC-PCR assay decreased the amount of time to final result compared to a cPCR assay.

Evaluation of the efficiency of nested q-PCR in the detection of Mycobacterium tuberculosis complex directly from tuberculosis-suspected lesions in post-mortem macroscopic inspections of bovine carcasses slaughtered in the state of Mato Grosso, Brazil.

PubMed

Carvalho, Ricardo César Tavares; Furlanetto, Leone Vinícius; Maruyama, Fernanda Harumy; Araújo, Cristina Pires de; Barros, Sílvia Letícia Bomfim; Ramos, Carlos Alberto do Nascimento; Dutra, Valéria; Araújo, Flábio Ribeiro de; Paschoalin, Vânia Margaret Flosi; Nakazato, Luciano; Figueiredo, Eduardo Eustáquio de Souza

2015-08-01

Bovine tuberculosis (BTB) is a zoonotic disease caused by Mycobacterium bovis, a member of the Mycobacterium tuberculosis complex (MTC). The quick and specific detection of this species is of extreme importance, since BTB may cause economic impacts, in addition to presenting imminent risks to human health. In the present study a nested real-time PCR test (nested q-PCR) was used in post-mortem evaluations to assess cattle carcasses with BTB-suspected lesions. A total of 41,193 cattle slaughtered in slaughterhouses located in the state of Mato Grosso, were examined. Of the examined animals, 198 (0.48%) showed BTB-suspected lesions. M. bovis was isolated in 1.5% (3/198) of the samples. Multiplex-PCR detected MTC in 7% (14/198) of the samples. The nested q-PCR test detected MTC in 28% (56/198) of the BTB-suspected lesions, demonstrating higher efficiency when compared to the multiplex-PCR and conventional microbiology. Nested q-PCR can therefore be used as a complementary test in the national program for control and eradication of bovine tuberculosis. Copyright © 2015 Elsevier Ltd. All rights reserved.

Evaluation of a Rapid Fecal PCR Test for Detection of Mycobacterium avium subsp. paratuberculosis in Dairy Cattle▿

PubMed Central

Wells, Scott J.; Collins, Michael T.; Faaberg, Kay S.; Wees, Carrie; Tavornpanich, Saraya; Petrini, Kristine R.; Collins, James E.; Cernicchiaro, Natalia; Whitlock, Robert H.

2006-01-01

A high-throughput TaqMan PCR assay for detection of bovine paratuberculosis was evaluated by using fecal samples from 1,808 dairy cattle in seven naturally infected herds and 347 dairy cattle in seven herds considered free of paratuberculosis. Fecal, blood, and milk samples were submitted to laboratories where the PCR-based assay, three different fecal culture procedures for Mycobacterium avium subsp. paratuberculosis (centrifugation, sedimentation, and the BACTEC filter concentration method), two serologic enzyme-linked immunosorbent assays (ELISAs), and one milk ELISA were performed. Results from testing of dairy cattle in herds free of M. avium subsp. paratuberculosis showed that the PCR assay's specificity was 99.7%. Twenty-three percent of the dairy cows that were fecal culture positive by at least one of the three methods were positive by the PCR assay. By Bayesian non-“gold standard” analysis methods, the TaqMan PCR assay had a higher specificity than the serum ELISAs (99.3%; 95% confidence interval [CI] = 98.6 to 99.7%) and a test sensitivity similar to that of the serum ELISAs (29%; 95% CI = 24 to 35%). By classical methods, the estimated relative sensitivity of the fecal PCR assay was 4% for light and moderate fecal shedders (compared to 12 to 13% for the ELISAs) and 76% for heavy fecal shedders (compared to 67% for the milk ELISA). The PCR assay has higher sensitivity for detection of heavy fecal shedders than the evaluated milk ELISA but lower sensitivity than a serum or milk ELISA for detection of light and moderate fecal shedders. This assay can be used as a quick test for detection of cattle with heavy fecal shedding, those cattle with the highest risk of transmitting infection to susceptible cattle. PMID:16928884

A nested polymerase chain reaction for the detection of genomic DNA of Myxobolus cerebralis in rainbow trout Oncorhynchus mykiss.

PubMed

Andree, K B; MacConnell, E; Hedrick, R P

1998-10-08

A nested polymerase chain reaction (PCR) test was developed to amplify a segment of the 18S rRNA gene from Myxobolus cerebralis, the agent causing whirling disease in salmonid fish. The PCR amplifies a 415 bp amplicon that was identified by dideoxynucleotide terminated sequencing to be identical to the known 18S rDNA sequence of M. cerebralis. There was no amplification of genomic DNA from 4 other myxosporean parasites of salmonid fish from the genus Myxobolus including M. arcticus, M. insidiosus, M. neurobius, and M. squamalis. The efficacy of the PCR test to detect early infections was demonstrated by amplification of the 415 bp fragment from experimentally exposed rainbow trout Oncorhynchus mykiss at 2 h and at 1, 2, and 3 wk postexposure to actinosporean stages (triactinomyxons) of M. cerebralis. In contrast, standard microscopic examinations of stained tissue sections of the same fish used for PCR were less reliable in detecting the presence of the parasite. Additional examinations of fish 5 mo postexposure, after sporogenesis had occurred, found the PCR to be a more reliable indicator of infection than pepsin-trypsin digest (PTD) method, particularly when trout were experimentally exposed to low levels of the infectious stages of the parasite. The PCR was able to amplify to detectable levels the equivalent of a single sporoplasm of M. cerebralis as found in a tissue sample. This test improves the detection of M. cerebralis because it can detect the presence of the parasite: (1) in both hosts, (2) in all known stages of its life cycle, and (3) at lower thresholds than currently used diagnostic methods. Lastly, the PCR test is less susceptible to morphological misidentifications of the spores that can occur with current microscopic procedures.

VIRUS ISOLATION AND MOLECULAR DETECTION OF BLUETONGUE AND EPIZOOTIC HEMORRHAGIC DISEASE VIRUSES FROM NATURALLY INFECTED WHITE-TAILED DEER (ODOCOILEUS VIRGINIANUS).

PubMed

Kienzle, Clara; Poulson, Rebecca L; Ruder, Mark G; Stallknecht, David E

2017-10-01

Hemorrhagic disease in North America is caused by multiple serotypes of epizootic hemorrhagic disease virus (EHDV) and bluetongue virus (BTV). Diagnostic tests for detection of EHDV and BTV include virus isolation (VI), reverse transcriptase (RT)-PCR, and real-time RT-PCR (rRT-PCR). Our objective was to compare the diagnostic capabilities of three rRT-PCR protocols for detection of EHDV and BTV from naturally infected white-tailed deer (Odocoileus virginianus). We compared the effectiveness of these assays to traditional viral detection methods (e.g., VI) for historic and current clinical cases. Because of the variable nature of tissue collection and storage before diagnostic testing, an evaluation of viral persistence on multiple freeze-thaw events was also conducted. Two of the rRT-PCR assays provided for reliable detection of EHDV and BTV from 100% of clinically affected and VI-confirmed infected animals. Additionally, no significant change in viral titer was observed on multiple freeze-thaw events.

A novel comprehensive set of fungal Real time PCR assays (fuPCR) for the detection of fungi in immunocompromised haematological patients-A pilot study.

PubMed

Rahn, Sebastian; Schuck, Anna; Kondakci, Mustafa; Haas, Rainer; Neuhausen, Nicole; Pfeffer, Klaus; Henrich, Birgit

2016-12-01

Fungal infections are recognized in an increasing number of patients with immunological deficits and are associated with high rates of mortality (Brown et al., 2012a). In this pilot-study, a rapid Real time PCR (fuPCR) was designed for the detection and differentiation of fungal pathogens in clinical specimens of haematological patients. The fuPCR, targeting the internal transcribed spacer region 2 (ITS2) of rDNA region, is comprised of seven multiplex reactions, which were shown to be specific and sensitive for a comprehensive spectrum of clinically relevant fungal species. This was validated by testing respective fungal DNAs in each fuPCR reaction and 28 respiratory samples of fungal pneumonia-proven patients. Clinical sample sets of throat swab, EDTA-blood and blood sera from 50 patients with severe haematological malignancies, including haematopoietic stem cell transfer (HSCT), and samples from 30 healthy individuals were then analysed. In a first step, 198 samples of immunosuppressed patients were solely examined by fuPCR; and 50.8% (33/65) respiratory swabs, 4.8% (3/63) EDTA blood samples and 1.4% (1/70) blood serum samples were tested positive. In a second step, 56 respiratory samples of immunosuppressed patients and 30 of healthy individuals were simultaneously analysed by fuPCR and standard cultivation techniques. By both methods 30.4% (17/56) swabs of the immunocompromised patients were tested positive, 37.5% (21/56) were tested negative and 32.1% (18/56) were tested fuPCR positive and culture negative. In analysing the blood samples of the immunocompromised patients 5.4% (3/56) EDTA blood samples and 16.1% (9/56) sera samples were tested fuPCR-positive, whereas all samples of 30 healthy individuals with no signs of immunological deficits were tested negative by fuPCR. 38.9% (14/36) of the fungi detected in respiratory samples of the immunosuppressed patients, belonged to Candida spp., 47.2% (17/36) to Saccharomyces spp., 5.6% (2/36) to Cladosporium spp. and 8.3% (3/36) to Alternaria spp., whereas cultivation only identified Candida spp. (10/17) and Saccharomyces spp. (7/17). In this pilot study a novel fuPCR assay was developed and validated for the simultaneous and comprehensive detection of fungal pathogens in clinical respiratory specimens of haematological patients. Future work will focus on the validation of the blood-stream detected fungi in pathogenicity of these patients. Copyright © 2016 Elsevier GmbH. All rights reserved.

Development of a pan-Simbu real-time reverse transcriptase PCR for the detection of Simbu serogroup viruses and comparison with SBV diagnostic PCR systems.

PubMed

Fischer, Melina; Schirrmeier, Horst; Wernike, Kerstin; Wegelt, Anne; Beer, Martin; Hoffmann, Bernd

2013-11-05

Schmallenberg virus (SBV), a novel orthobunyavirus of the Simbu serogroup, was first identified in October 2011 in dairy cattle in Germany, where it caused fever, diarrhea and a drop in milk yield. Since then, SBV additionally has been detected in adult sheep and goats. Although symptoms of acute infection were not observed, infection during a vulnerable phase of pregnancy caused congenital malformations and stillbirths. In view of the current situation and the possible emergence of further Simbu serogroup members, a pan-Simbu real-time reverse transcriptase (RT) PCR system for the reliable detection of Simbu serogroup viruses should be developed. In this study a pan-Simbu real-time RT-PCR system was established and compared to several SBV real-time RT-PCR assays. All PCR-systems were tested using a panel of different Simbu serogroup viruses as well as several field samples from diseased cattle, sheep and goats originating from all over Germany. Several pan-Simbu real-time RT-PCR products were sequenced via Sanger sequencing. Furthermore, in silico analyses were performed to investigate suitability for the detection of further orthobunyaviruses. All tested members of the Simbu serogroup (n = 14) as well as most of the field samples were successfully detected by the pan-Simbu real-time RT-PCR system. The comparison of this intercalating dye assay with different TaqMan probe-based assays developed for SBV diagnostics confirmed the functionality of the pan-Simbu assay for screening purposes. However, the SBV-TaqMan-assay SBV-S3 delivered the highest analytical sensitivity of less than ten copies per reaction for duplex systems including an internal control. In addition, for confirmation of SBV-genome detection the highly specific SBV-M1 assay was established. The pan-Simbu real-time RT-PCR system was able to detect all tested members of the Simbu serogroup, most of the SBV field samples as well as three tested Bunyamwera serogroup viruses with a suitable sensitivity. According to in silico analyses, this system seems to be able to detect a broad orthobunyavirus spectrum. As an additional feature of the pan-Simbu real-time RT-PCR system, subsequent species classification via sequencing is feasible. Regarding SBV diagnostics, the performance of the S-segment targeting SBV-S3 assay was superior with respect to the analytical sensitivity.

An international inter-laboratory ring trial to evaluate a real-time PCR assay for the detection of bovine herpesvirus 1 in extended bovine semen.

PubMed

Wang, Jianning; O'Keefe, Joseph; Orr, Della; Loth, Leo; Banks, Malcolm; Wakeley, Philip; West, Donna; Card, Roderick; Ibata, Georgina; Van Maanen, Kees; Thoren, Peter; Isaksson, Mats; Kerkhofs, Pierre

2008-01-01

Six laboratories participated in a ring trial to evaluate the reliability of a real-time PCR assay for the detection of bovine herpesvirus 1 (BoHV-1) from extended bovine semen. Sets of coded samples were prepared and distributed to each of the laboratories. The sample panel contained semen from naturally and artificially infected bulls, serial dilutions of positive semen with negative semen, semen from uninfected seronegative bulls, negative semen spiked with virus, as well as serial dilutions of reference virus. The samples were tested using a previously validated real-time PCR assay for the detection of BoHV-1 in each participating laboratory. The PCR tests were conducted with four different real-time PCR amplification platforms, including RotorGene 3000, Stratagene MX 3000/4000, ABI 7900, and Roche LightCycler 2.0. Virus isolation using one set of samples was performed in one laboratory. The results of the laboratories were compared with one another, and with those of virus isolation. It was found that the sensitivity and specificity of the real-time PCR test was greater than those of virus isolation (82.7% versus 53.6% and 93.6% versus 84.6%, respectively). A high level of agreement on PCR testing results between the laboratories was achieved (kappa value 0.59-0.95). The results of this study indicate that the real-time PCR assay is suitable for the detection of BoHV-1 in extended semen, and would be a good substitute for the slow and laborious virus isolation, for the screening testing at artificial insemination centres and for international trade.

Prevalence of Vector-Borne Pathogens in Southern California Dogs With Clinical and Laboratory Abnormalities Consistent With Immune-Mediated Disease.

PubMed

Kidd, L; Qurollo, B; Lappin, M; Richter, K; Hart, J R; Hill, S; Osmond, C; Breitschwerdt, E B

2017-07-01

Studies investigating the prevalence of vector-borne pathogens in southern California dogs are limited. Occult infections might be misdiagnosed as idiopathic immune-mediated disease. (1) To determine the prevalence of vector-borne pathogens in southern California dogs with compatible clinical findings using PCR and serologic panels and (2) to determine whether testing convalescent samples and repeating PCR on acute samples using the same and different gene targets enhance detection. Forty-two client-owned dogs with clinical signs of vector-borne disease presenting to specialty practices in San Diego County. Combined prospective and retrospective observational study. Forty-two acute and 27 convalescent samples were collected. Acute samples were prospectively tested for antibodies to Rickettsia, Ehrlichia, Bartonella, Babesia, Borrelia, and Anaplasma species. PCR targeting Ehrlichia, Babesia, Anaplasma, hemotropic Mycoplasma, and Bartonella species was also performed. Retrospectively, convalescent samples were tested for the same organisms using serology, and for Ehrlichia, Babesia, Anaplasma, and Bartonella species using PCR. Acute samples were retested using PCR targeting Ehrlichia and Babesia species. Evidence of exposure to or infection with a vector-borne pathogen was detected in 33% (14/42) of dogs. Ehrlichia and Babesia species were most common; each was identified in 5 dogs. Convalescent serologic testing, repeating PCR, and using novel PCR gene targets increased detection by 30%. Repeated testing using serology and PCR enhances detection of infection by vector-borne pathogens in dogs with clinical signs of immune-mediated disease. Larger prevalence studies of emerging vector-borne pathogens in southern California dogs are warranted. Copyright © 2017 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals, Inc. on behalf of the American College of Veterinary Internal Medicine.

Detection of Trypanosoma cruzi infection in naturally-infected dogs and cats using serological, parasitological and molecular methods

PubMed Central

Enriquez, G.F.; Cardinal, M.V.; Orozco, M.M.; Schijman, A.G.; Gürtler, R.E.

2013-01-01

Domestic dogs and cats are major domestic reservoir hosts of Trypanosoma cruzi and a risk factor for parasite transmission. In this study we assessed the relative performance of a polymerase chain reaction assay targeted to minicircle DNA (kDNA-PCR) in reference to conventional serological tests, a rapid dipstick test and xenodiagnosis to detect T. cruzi infection in dogs and cats from an endemic rural area in northeastern Argentina. A total of 43 dogs and 13 cats seropositive for T. cruzi by an immunosorbent assay (ELISA) and an indirect hemagglutination assay (IHA), which had been examined by xenodiagnosis, were also tested by kDNA-PCR. kDNA-PCR was nearly as sensitive as xenodiagnosis for detecting T. cruzi- infectious dogs and cats. kDNA-PCR was slightly more sensitive than xenodiagnosis in seropositive dogs (91% versus 86%, respectively) and cats (77% against 54%, respectively), but failed to detect all of the seropositive individuals. ELISA and IHA detected all xenodiagnosis-positive dogs and both outcomes largely agreed (kappa coefficient, κ = 0.92), whereas both assays failed to detect all of the xenodiagnosis-positive cats and their agreement was moderate (κ = 0.68). In dogs, the sensitivity of the dipstick test was 95% and agreed closely with the outcome of conventional serological tests (κ = 0.82). The high sensitivity of kDNA-PCR to detect T. cruzi infections in naturally-infected dogs and cats supports its application as a diagnostic tool complementary to serology and may replace the use of xenodiagnosis or hemoculture. PMID:23499860

Development of real-time PCR tests for the detection of Tenebrio molitor in food and feed.

PubMed

Debode, Frédéric; Marien, Aline; Gérard, Amaury; Francis, Frédéric; Fumière, Olivier; Berben, Gilbert

2017-08-01

Insects are rich in proteins and could be an alternative source of proteins to feed animals and humans. Numerous companies have started the production of insects for feed purposes. In Europe, these processed animal proteins are not yet authorised by legislation as many questions still need to be answered concerning this 'novel food'. Authorisations will be possible when methods of authentication of the products are available. In this study we propose real-time PCR methods for the specific detection of the mealworm (Tenebriomolitor), one of the most widely used insects for food and feed production. Two PCR assays are proposed: the first based on the wingless gene and the second based on the cadherin gene. The PCR tests amplify fragments of 87 bp. These qualitative methods were tested according to several performance criteria. The specificity was tested on 34 insect species' DNA, but also on non-insect species including crustacean, mammals, birds and plants. The limit of detection was determined and was below 20 copies for the two PCR tests. The applicability of the tests was demonstrated by the analysis of real-life processed samples containing T. molitor.

Detection and Species Determination of Malaria Parasites by PCR: Comparison with Microscopy and with ParaSight-F and ICT Malaria Pf Tests in a Clinical Environment

PubMed Central

Tham, Jill M.; Lee, Szu Hee; Tan, Theresa M. C.; Ting, Robert C. Y.; Kara, Ursula A. K.

1999-01-01

A rapid procedure for the diagnosis of malaria infections directly from dried blood spots by PCR amplification was evaluated with samples from 52 patients. Plasmodium infections were identified with a genus-specific primer set, and species differentiation between Plasmodium falciparum and Plasmodium vivax was analyzed by multiplex PCR. The PCR test with any of the three primer sets was able to detect as few as four parasites per microliter by gel electrophoresis or by nonisotopic paper hybridization chromatography. The diagnoses obtained by PCR correlated closely with those obtained by Giemsa staining except for two samples observed to have mixed P. falciparum-P. vivax infections. These were initially missed by microscopic analysis. In comparison with antigen-capture assays for P. falciparum, the PCR assays were able to detect three infections that were missed by the ParaSight-F test. The PCR test was negative for nine ParaSight-F-positive samples and one ICT Malaria Pf-positive sample, and these were confirmed to be false-positive results. The PCR thus gave no false-negative or false-positive results. Patients undergoing antimalarial therapy were also monitored by the PCR assay. Four of seven patients who were PCR positive for P. vivax at the time of discharge were later readmitted to the hospital with a recurrence of P. vivax infection. We would like to propose that PCR is a sensitive and easy method that can serve as a useful addition to microscopy for the diagnosis and the clinical monitoring of treatment of malaria. PMID:10203469

Porphyromonas gingivalis, Porphyromonas endodontalis, Prevotella intermedia and Prevotella nigrescens in endodontic lesions detected by culture and by PCR.

PubMed

Gomes, B P F A; Jacinto, R C; Pinheiro, E T; Sousa, E L R; Zaia, A A; Ferraz, C C R; Souza-Filho, F J

2005-08-01

he aim of this study was to investigate the presence of four black-pigmented bacteria, Porphyromonas gingivalis, Porphyromonas endodontalis, Prevotella intermedia and Prevotella nigrescens, in endodontic infections by culture and polymerase chain reaction (PCR) analyses. Microbial samples were obtained from 50 teeth with untreated necrotic pulps (primary infection) and from 50 teeth with failing endodontic treatment (secondary infection). Microbiological strict anaerobic techniques were used for serial dilution, plating, incubation, and identification. For PCR detection, the samples were analyzed using species-specific primers of 16S rDNA and the downstream intergenic spacer region. Culture and PCR detected the test species in 13/100 and 50/100 of the study teeth, respectively. The organisms were cultured from 11/50 (22%) of primarily infected root canal samples and from 2/50 (4%) of secondary root canal samples. PCR detection identified the target species in 32/50 (64%) and 18/50 (36%) of primary and secondary infections, respectively. P. gingivalis was rarely isolated by culture methods (1%), but was the most frequently identified test species by PCR (38%). Similarly, P. endodontalis was not recovered by culture from any tooth studied, but was detected by PCR in 25% of the sampled teeth. PCR-based identification also showed higher detection rates of P. intermedia (33%) and P. nigrescens (22%) than culture (13%). In conclusion, P. gingivalis, P. endodontalis, P. intermedia, and P. nigrescens were identified more frequently in teeth with necrotic pulp than in teeth with failing endodontic treatment. Also, a higher frequency of black-pigmented species was detected by PCR than by culture.

Validation of the Pockit Dengue Virus Reagent Set for Rapid Detection of Dengue Virus in Human Serum on a Field-Deployable PCR System.

PubMed

Tsai, Jih-Jin; Liu, Li-Teh; Lin, Ping-Chang; Tsai, Ching-Yi; Chou, Pin-Hsing; Tsai, Yun-Long; Chang, Hsiao-Fen Grace; Lee, Pei-Yu Alison

2018-05-01

Dengue virus (DENV) infection, a mosquito-borne disease, is a major public health problem in tropical countries. Point-of-care DENV detection with good sensitivity and specificity enables timely early diagnosis of DENV infection, facilitating effective disease management and control, particularly in regions of low resources. The Pockit dengue virus reagent set (GeneReach Biotech), a reverse transcription insulated isothermal PCR (RT-iiPCR), is available to detect all four serotypes of DENV on the field-deployable Pockit system, which is ready for on-site applications. In this study, analytical and clinical performances of the assay were evaluated. The index assay did not react with 14 non-DENV human viruses, indicating good specificity. Compared to the U.S. CDC DENV-1-4 real-time quantitative RT-PCR (qRT-PCR) assay, testing with serial dilutions of virus-spiked human sera demonstrated that the index assay had detection endpoints that were separately comparable with the 4 serotypes. Excellent reproducibility was observed among repeat tests done by six operators at three sites. In clinical performance, 195 clinical sera collected around Kaohsiung city in 2012 and 21 DENV-4-spiked sera were tested with the RT-iiPCR and qRT-PCR assays in parallel. The 121 (11 DENV-1, 78 DENV-2, 11 DENV-3, and 21 DENV-4) qRT-PCR-positive and 95 qRT-PCR-negative samples were all positive and negative by the RT-iiPCR reagent results, respectively, demonstrating high (100%) interrater agreement (95% confidence interval [CI 95% ], ∼98.81% to 100%; κ = 1). With analytical and clinical performance equivalent to those of the reference qRT-PCR assay, the index PCR assay on the field-deployable system can serve as a highly sensitive and specific on-site tool for DENV detection. Copyright © 2018 American Society for Microbiology.

Usefulness of multiplex PCR methods and respiratory viruses’ distribution in children below 15 years old according to age, seasons and clinical units in France: A 3 years retrospective study

PubMed Central

Collin, Gilles; Ichou, Houria; Charpentier, Charlotte; Bendhafer, Samia; Dumitrescu, Madalina; Allal, Lahcene; Cojocaru, Bogdan; Desfrère, Luc; Descamps, Diane; Mandelbrot, Laurent; Houhou-Fidouh, Nadhira

2017-01-01

Background To date, only influenza and RSV testing are recommended for respiratory viruses’ detection in paediatric units. In this study, we described, according to seasons, ages and clinical units, the results obtained in children (<15 years old) by multiplex-PCR (mPCR) tests allowing a quick and wide range detection of all respiratory viruses. These results were also compared with RSV specific detection. Methods All nasopharyngeal mPCR and RSV tests requested by clinicians in our French teaching hospitals group between 2011 and 2014 were retrospectively included. All repeated samples for the same children in the same month were discarded. Results Of the 381 mPCR tests (344 children) performed, 51.4% were positive. Positivity and viral co-infection rates were higher in the 6–36 months old strata (81% and 25%, p<0.0001 and p = 0.04, respectively). Viral distribution showed strong variations across ages. During specific influenza epidemic periods, only 1/39 (2.5%) mPCR tests were positive for influenza and 19/39 (48.7%) for other viruses. During specific RSV epidemic periods, only 8/46 (17.4%) mPCR tests were positive for RSV and 14/46 (30.4%) for other viruses. 477/1529 (31.2%) of RSV immunochromatography-tests were positive. Among the negatives immunochromatography-test also explored by mPCR, 28/62 (31%) were positive for other respiratory viruses. Conclusion This study provides a wide description of respiratory viruses’ distribution among children in hospital settings using mPCR over 3 years. It emphasizes the number of undiagnosed respiratory viruses according to the current diagnosis practice in France and gives a better picture of respiratory viruses identified in hospital settings by mPCR all over the year in France. PMID:28235002

[Correlation between results of PCR and specific serological tests in diagnosis of Epstein-Barr virus in patients with mononucleosis syndrome].

PubMed

Banko, A V; Lazarević, I B; Cupić, M D; Knezević, A M; Stevanović, G D; Krejović-Trivić, S B; Jovanović, T P

2009-01-01

Routine laboratory diagnosis of infectious mononucleosis is based on EBV serological testing, but due to problems in interpretation of results, molecular methods, especially PCR, are often necessary. The aim of the present study was to investigate correlation between results of PCR and specific serological tests in diagnosis of Epstein-Barr virus in patients with mononucleosis syndrome. The study comprised 68 patients with mononucleosis syndrome. Their blood samples were tested using ELISA for detection of 4 EBV specific antibodies (anti-VCA IgM and IgG, anti-EA-D IgG and anti-EBNA-1 IgG) and PCR for detection of EBV DNA. According to results of serology 42 patients had acute primary infection, 2 reactivation, 1 chronic active infection, 19 past infection, and 4 have been EBV seronegative. EBV DNA was detected in 17 patients (25%) and all of them were serologically defined as acutely infected. PCR was useful for resolving unclear serology results. Specific serology is the first step in diagnosis of IM, but PCR may serve as a useful additional diagnostic tool for clarifying serological dilemmas, reaching final diagnosis and defining status of the infection.

Detection of Dirofilaria immitis and other arthropod-borne filarioids by an HRM real-time qPCR, blood-concentrating techniques and a serological assay in dogs from Costa Rica.

PubMed

Rojas, Alicia; Rojas, Diana; Montenegro, Víctor M; Baneth, Gad

2015-03-23

Canine filarioids are important nematodes transmitted to dogs by arthropods. Diagnosis of canine filariosis is accomplished by the microscopic identification of microfilariae, serology or PCR for filarial-DNA. The aim of this study was to evaluate a molecular assay for the detection of canine filariae in dog blood, to compare its performance to other diagnostic techniques, and to determine the relationship between microfilarial concentration and infection with other vector-borne pathogens. Blood samples from 146 dogs from Costa Rica were subjected to the detection of canine filarioids by four different methods: the microhematocrit tube test (MCT), Knott's modified test, serology and a high resolution melt and quantitative real-time PCR (HRM-qPCR). Co-infection with other vector-borne pathogens was also evaluated. Fifteen percent of the dogs were positive to Dirofilaria immitis by at least one of the methods. The HRM-qPCR produced distinctive melting plots for the different filarial worms and revealed that 11.6% of dogs were infected with Acanthocheilonema reconditum. The latter assay had a limit of detection of 2.4x10⁻⁴ mf/μl and detected infections with lower microfilarial concentrations in comparison to the microscopic techniques and the serological assay. The MCT and Knott's test only detected dogs with D. immitis microfilaremias above 0.7 mf/μl. Nevertheless, there was a strong correlation between the microfilarial concentration obtained by the Knott's modified test and the HRM-qPCR (r = 0.906, p < 0.0001). Interestingly, one dog was found infected with Cercopithifilaria bainae infection. Moreover, no association was found between microfilaremia and co-infection and there was no significant difference in microfilarial concentration between dogs infected only with D. immitis and dogs co-infected with Ehrlichia canis, Anaplasma platys or Babesia vogeli. This is the first report of A. reconditum and C. bainae in Costa Rica and Central America. Among the evaluated diagnostic techniques, the HRM-qPCR showed the most sensitive and reliable performance in the detection of blood filaroids in comparison to the Knott's modified test, the MCT test and a serological assay.

A New Single-Step PCR Assay for the Detection of the Zoonotic Malaria Parasite Plasmodium knowlesi

PubMed Central

Lucchi, Naomi W.; Poorak, Mitra; Oberstaller, Jenna; DeBarry, Jeremy; Srinivasamoorthy, Ganesh; Goldman, Ira; Xayavong, Maniphet; da Silva, Alexandre J.; Peterson, David S.; Barnwell, John W.; Kissinger, Jessica; Udhayakumar, Venkatachalam

2012-01-01

Background Recent studies in Southeast Asia have demonstrated substantial zoonotic transmission of Plasmodium knowlesi to humans. Microscopically, P. knowlesi exhibits several stage-dependent morphological similarities to P. malariae and P. falciparum. These similarities often lead to misdiagnosis of P. knowlesi as either P. malariae or P. falciparum and PCR-based molecular diagnostic tests are required to accurately detect P. knowlesi in humans. The most commonly used PCR test has been found to give false positive results, especially with a proportion of P. vivax isolates. To address the need for more sensitive and specific diagnostic tests for the accurate diagnosis of P. knowlesi, we report development of a new single-step PCR assay that uses novel genomic targets to accurately detect this infection. Methodology and Significant Findings We have developed a bioinformatics approach to search the available malaria parasite genome database for the identification of suitable DNA sequences relevant for molecular diagnostic tests. Using this approach, we have identified multi-copy DNA sequences distributed in the P. knowlesi genome. We designed and tested several novel primers specific to new target sequences in a single-tube, non-nested PCR assay and identified one set of primers that accurately detects P. knowlesi. We show that this primer set has 100% specificity for the detection of P. knowlesi using three different strains (Nuri, H, and Hackeri), and one human case of malaria caused by P. knowlesi. This test did not show cross reactivity with any of the four human malaria parasite species including 11 different strains of P. vivax as well as 5 additional species of simian malaria parasites. Conclusions The new PCR assay based on novel P. knowlesi genomic sequence targets was able to accurately detect P. knowlesi. Additional laboratory and field-based testing of this assay will be necessary to further validate its utility for clinical diagnosis of P. knowlesi. PMID:22363751

What assay is optimal for the diagnosis of measles virus infection? An evaluation of the performance of a measles virus real-time reverse transcriptase PCR using the Cepheid SmartCycler(®) and antigen detection by immunofluorescence.

PubMed

Chua, Kyra Y L; Thapa, Kiran; Yapa, Chaturangi M; Somerville, Lucy K; Chen, Sharon C-A; Dwyer, Dominic E; Sheppeard, Vicky; Kok, Jen

2015-09-01

Despite the World Health Organization (WHO)-reported elimination of measles in Australia, importation of cases especially in travellers from Asia continues in Sydney, Australia's largest city. Laboratory confirmation supports clinico-epidemiological evidence of measles virus infection, and is needed to establish elimination. To evaluate the performance of a random access real-time reverse transcriptase polymerase chain reaction (RT-PCR) assay using the moderate complexity SmartCycler(®) platform, and measles antigen detection by immunofluorescence (IFA), for the detection of measles virus in patient samples. One hundred samples comprising nose and throat swabs, nasopharyngeal aspirates and urine, collected from patients with suspected measles were tested in parallel using IFA and nucleic acid testing using the SmartCycler(®) and LightCycler(®) RT-PCR platforms. The LightCycler(®) RT-PCR was used as the reference assay against which the SmartCycler(®) RT-PCR and IFA were compared. Using the LightCycler(®) RT-PCR, measles virus was detected in 35 clinical samples. There was 100% concordance between the results of the SmartCycler(®) and the LightCycler(®)-based RT-PCR. Measles genotypes detected included B3, D8, and D9. Testing urine in addition to NTS did not improve diagnostic yield. In contrast, the sensitivity and specificity of IFA compared to the reference LightCycler(®) RT-PCR was 34.3% and 96.7%, respectively. The performance of the SmartCycler(®) is comparable to the LightCycler(®) for the detection of measles virus. However, IFA had poor sensitivity and should not be used to confirm measles virus infection where nucleic acid testing is available. Copyright © 2015 Elsevier B.V. All rights reserved.

Comparison of ELISA and RT-PCR for the detection of Prunus necrotic ring spot virus and prune dwarf virus in almond (Prunus dulcis).

PubMed

Mekuria, Genet; Ramesh, Sunita A; Alberts, Evita; Bertozzi, Terry; Wirthensohn, Michelle; Collins, Graham; Sedgley, Margaret

2003-12-01

A technique based on the reverse transcriptase-polymerase chain reaction (RT-PCR) has been developed to detect the presence of Prunus necrotic ringspot virus (PNRSV) and prune dwarf virus (PDV) simultaneously in almond. This paper presents the results of a 3-year study comparing both enzyme-linked immunosorbent assay (ELISA) and RT-PCR for the detection of PNRSV and PDV using 175 almond leaf samples. Multiplex RT-PCR was found to be more sensitive than ELISA, especially when followed by nested PCR for the detection of PDV. The RT-PCR technique has the added advantage that plant material can be tested at any time throughout the growing season.

Detection of Hepatitis B Virus DNA among Chronic and potential Occult HBV patients in resource-limited settings by Loop-Mediated Isothermal Amplification assay.

PubMed

Akram, Arifa; Islam, S M Rashedul; Munshi, Saif Ullah; Tabassum, Shahina

2018-05-16

Transmission of Hepatitis B Virus (HBV) usually occurs due to the transfusion of blood or blood products from chronic HBV (CHB) or occult HBV infected (OBI) patients. Besides serological tests e.g. HBsAg and anti-HBc (total), detection of HBV-DNA is necessary for the diagnosis of OBI patients. Different nucleic acid tests (NATs) including real-time-Polymerase Chain Reaction (qPCR) are used for the detect HBV-DNA. The NATs are expensive and require technical expertise which are barriers to introducing them in resource-limited settings. This study was undertaken to evaluate the use of Loop-Mediated Isothermal Amplification (LAMP) assay as an alternative to qPCR for the detection of HBV-DNA in CHB and potential OBI patients in resource-limited settings. Following the published protocols with some modifications, a LAMP assay was developed for detection of HBV-DNA by either using a heat block followed by detection in an agarose gel or using a qPCR thermocycler. The LAMP assay was applied to supernatant prepared from heat treated serum collected from CHB and potential OBI patients. HBV viral load in serum was measured by qPCR using a single step HBV-DNA quantification kit. Among 200 samples tested, qPCR was capable to detect HBV-DNA in 25.5% of cases, whereas LAMP assay detected HBV-DNA in 43.5% cases. The qPCR was able to detect 11 (9.16%) potential OBI cases, whereas LAMP assay identified HBV-DNA in 43 (35.83%) cases. In addition to tests for HBsAg and/or anti-HBc (total), detection of HBV-DNA by LAMP assay may aid in preventing post-transfusion HBV infection in resource-limited settings. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

The physiologic state of Escherichia coli O157:H7 does not affect its detection in two commercial real-time PCR-based tests

USDA-ARS?s Scientific Manuscript database

Multiplex real-time PCR detection of Escherichia coli O157:H7 is an efficient molecular tool with high sensitivity and specificity for meat safety and quality assurance in the beef industry. The Biocontrol GDS and the DuPont Qualicon BAX®-RT rapid detection systems are two commercial tests based on...

Evaluation of efficiency of nested multiplex allele-specific PCR assay for detection of multidrug resistant tuberculosis directly from sputum samples.

PubMed

Mistri, S K; Sultana, M; Kamal, S M M; Alam, M M; Irin, F; Nessa, J; Ahsan, C R; Yasmin, M

2016-05-01

For an effective control of tuberculosis, rapid detection of multidrug resistant tuberculosis (MDR-TB) is necessary. Therefore, we developed a modified nested multiplex allele-specific polymerase chain reaction (MAS-PCR) method that enables rapid MDR-TB detection directly from sputum samples. The efficacy of this method was evaluated using 79 sputum samples collected from suspected tuberculosis patients. The performance of nested MAS-PCR method was compared with other MDR-TB detection methods like drug susceptibility testing (DST) and DNA sequencing. As rifampicin (RIF) resistance conforms to MDR-TB in greater than 90% cases, only the presence of RIF-associated mutations in rpoB gene was determined by DNA sequencing and nested MAS-PCR to detect MDR-TB. The concordance between nested MAS-PCR and DNA sequencing results was found to be 96·3%. When compared with DST, the sensitivity and specificity of nested MAS-PCR for RIF-resistance detection were determined to be 92·9 and 100% respectively. For developing- and high-TB burden countries, molecular-based tests have been recommended by the World Health Organization for rapid detection of MDR-TB. The results of this study indicate that, nested MAS-PCR assay might be a practical and relatively cost effective molecular method for rapid detection of MDR-TB from suspected sputum samples in developing countries with resource poor settings. © 2016 The Society for Applied Microbiology.

Rapid detection methods for viable Mycobacterium avium subspecies paratuberculosis in milk and cheese.

PubMed

Botsaris, George; Slana, Iva; Liapi, Maria; Dodd, Christine; Economides, Constantinos; Rees, Catherine; Pavlik, Ivo

2010-07-31

Mycobacterium avium subsp. paratuberculosis (MAP) may have a role in the development of Crohn's disease in humans via the consumption of contaminated milk and milk products. Detection of MAP from milk and dairy products has been reported from countries on the European continent, Argentina, the UK and Australia. In this study three different methods (quantitative real time PCR, combined phage IS900 PCR and conventional cultivation) were used to detect the presence of MAP in bulk tank milk (BTM) and cheese originating from sheep, goat and mixed milks from farms and products in Cyprus. During the first survey the presence of MAP was detected in 63 (28.6%) of cows' BTM samples by quantitative real time PCR. A second survey of BTM used a new combined phage IS900 PCR assay, and in this case MAP was detected in 50 (22.2%) samples showing a good level of agreement by both methods. None of the herds tested were known to be affected by Johne's disease and the presence of viable MAP was confirmed by conventional culture in only two cases of cows BTM. This suggests that either rapid method used is more sensitive than the conventional culture when testing raw milk samples for MAP. The two isolates recovered from BTM were identified by IS1311 PCR REA as cattle and sheep strains, respectively. In contrast when cheese samples were tested, MAP DNA was detected by quantitative real time PCR in seven (25.0%) samples (n=28). However no viable MAP was detected when either the combined phage IS900 PCR or conventional culture methods were used. Copyright 2010 Elsevier B.V. All rights reserved.

Comparison between two commercially available serological tests and polymerase chain reaction in the diagnosis of Cryptosporidium in animals and diarrhoeic children.

PubMed

Helmy, Yosra A; Krücken, Jürgen; Nöckler, Karsten; von Samson-Himmelstjerna, Georg; Zessin, Karl-H

2014-01-01

For the detection of Cryptosporidium species in 804 animals and 165 diarrhoeic children (<10 years) in Egypt, two copro-antigen tests, the RIDASCREEN® Cryptosporidium test [enzyme immunoassay (EIA)] and the RIDA®QUICK Cryptosporidium/Giardia Combi [immuno-chromatographic test (ICT)] as well as polymerase chain reaction (PCR) were used. Prevalence of Cryptosporidium was 15.0, 19.5 and 32.3% in animals and 2.4, 6.7 and 49.1% in children using EIA, ICT and PCR, respectively.Using PCR as reference method, animal samples sensitivity (Se) of the EIA was 46.5% when questionable samples were considered positive, whereas specificity (Sp) was 100%. Se of the ICT was 60.4% while Sp was 100%. Positive predictive values (PPVs) for both EIA and ICT test were 100%, and negative predictive values (NPVs) for EIA were 79.7 and 84.1% for ICT. For the children samples, the Se of EIA was 5%, Sp was 100%, PPV was 100% and NPV was 52.2%, while the Se of ICT was 13.6%, Sp was 100%, PPV was 100% and NPV was 54.6%.The Kappa score of agreement between PCR and ICT was 67.4%, 54.1% between PCR and EIA and 84.4% between ICT and EIA. Until the second serial dilution of the EIA and ICT test, 9 × 10(3) oocysts/μl of Cryptosporidia was detected, whereas in PCR, they were detected until the sixth serial dilution. Copro-antigen tests were easy to perform and less time-consuming but less sensitive compared to PCR. They obviously are best applicable for screening and epidemiological studies of large numbers of subjects, for batch specimen processing and in isolated or rural areas where reliable tests like PCR are unfeasible. When in children, a single stool sample is used for the diagnosis of clinical cases; better results can be obtained when non-standardized PCR due low specificity is coupled with copro-antigen tests.

Comparison of real-time PCR with disk diffusion, agar screen and E-test methods for detection of methicillin-resistant Staphylococcus aureus.

PubMed

Shariati, Laleh; Validi, Majid; Tabatabaiefar, Mohammad Amin; Karimi, Ali; Nafisi, Mohammad Reza

2010-12-01

Methicillin-resistant Staphylococcus aureus (MRSA) is a nosocomial pathogen. Our main objective was to compare oxacillin disk test, oxacillin E-test, and oxacillin agar screen for detection of methicillin resistance in S. aureus, using real-time PCR for mecA as the "gold standard" comparison assay. 196 S. aureus isolates were identified out of 284 Staphylococcus isolates. These isolates were screened for MRSA with several methods: disk diffusion, agar screen (6.0 μg/ml), oxacillin E-test, and real-time PCR for detection of mecA gene. Of the 196 S. aureus isolates tested, 96 isolates (49%) were mecA-positive and 100 isolates (51%) mecA-negative. All methods tested had a statistically significant agreement with real-time PCR. E-test was 100% sensitive and specific for mecA presence. The sensitivity and specificity of oxacillin agar screen method were 98 and 99%, respectively and sensitivity and specificity of oxacillin disk diffusion method were 95 and 93%, respectively. In the present study, oxacillin E-test is proposed as the best phenotypic method. For economic reasons, the oxacillin agar screen method (6.0 μg/ml), which is suitable for the detection of MRSA, is recommended due to its accuracy and low cost.

Evaluation of Aspergillus PCR protocols for testing serum specimens.

PubMed

White, P Lewis; Mengoli, Carlo; Bretagne, Stéphane; Cuenca-Estrella, Manuel; Finnstrom, Niklas; Klingspor, Lena; Melchers, Willem J G; McCulloch, Elaine; Barnes, Rosemary A; Donnelly, J Peter; Loeffler, Juergen

2011-11-01

A panel of human serum samples spiked with various amounts of Aspergillus fumigatus genomic DNA was distributed to 23 centers within the European Aspergillus PCR Initiative to determine analytical performance of PCR. Information regarding specific methodological components and PCR performance was requested. The information provided was made anonymous, and meta-regression analysis was performed to determine any procedural factors that significantly altered PCR performance. Ninety-seven percent of protocols were able to detect a threshold of 10 genomes/ml on at least one occasion, with 83% of protocols reproducibly detecting this concentration. Sensitivity and specificity were 86.1% and 93.6%, respectively. Positive associations between sensitivity and the use of larger sample volumes, an internal control PCR, and PCR targeting the internal transcribed spacer (ITS) region were shown. Negative associations between sensitivity and the use of larger elution volumes (≥100 μl) and PCR targeting the mitochondrial genes were demonstrated. Most Aspergillus PCR protocols used to test serum generate satisfactory analytical performance. Testing serum requires less standardization, and the specific recommendations shown in this article will only improve performance.

Evaluation of Aspergillus PCR Protocols for Testing Serum Specimens▿†

PubMed Central

White, P. Lewis; Mengoli, Carlo; Bretagne, Stéphane; Cuenca-Estrella, Manuel; Finnstrom, Niklas; Klingspor, Lena; Melchers, Willem J. G.; McCulloch, Elaine; Barnes, Rosemary A.; Donnelly, J. Peter; Loeffler, Juergen

2011-01-01

A panel of human serum samples spiked with various amounts of Aspergillus fumigatus genomic DNA was distributed to 23 centers within the European Aspergillus PCR Initiative to determine analytical performance of PCR. Information regarding specific methodological components and PCR performance was requested. The information provided was made anonymous, and meta-regression analysis was performed to determine any procedural factors that significantly altered PCR performance. Ninety-seven percent of protocols were able to detect a threshold of 10 genomes/ml on at least one occasion, with 83% of protocols reproducibly detecting this concentration. Sensitivity and specificity were 86.1% and 93.6%, respectively. Positive associations between sensitivity and the use of larger sample volumes, an internal control PCR, and PCR targeting the internal transcribed spacer (ITS) region were shown. Negative associations between sensitivity and the use of larger elution volumes (≥100 μl) and PCR targeting the mitochondrial genes were demonstrated. Most Aspergillus PCR protocols used to test serum generate satisfactory analytical performance. Testing serum requires less standardization, and the specific recommendations shown in this article will only improve performance. PMID:21940479

Performance of polymerase chain reaction for the diagnosis of cystic echinococcosis using serum, urine, and cyst fluid samples

PubMed Central

Chaya, DR; Parija, Subhash Chandra

2014-01-01

Introduction: Cystic echinococcosis (CE) is a chronic zoonosis which presents with variable clinical manifestations. Currently the diagnosis of this disease is based on radiological findings and serological tests which lack specificity. Although antigen detection from the cyst fluid is the most specific, it is seldom done due to the complications involved. Detecting the presence of Echinococcus granulosus specific deoxyribonucleic acid (DNA) by the polymerase chain reaction (PCR) could provide a definitive diagnosis of CE. Materials and Methods: An in-house PCR assay was devised to detect E. granulosus specific DNA in serum, urine and hydatid cyst fluid. The ability of the PCR to detect E. granulosus in the above mentioned samples were observed in comparison with other antigen and antibody detection tests. Results: Serum samples from surgically confirmed patients of CE with ruptured cysts contained the corresponding DNA while the in the majority of cases who had an intact cyst had no DNA of E. granulosus in their serum. DNA of E. granulosus was not found to be excreted in urine. PCR performed equal to antigen detection ELISA while testing hydatid cyst fluid samples. Conclusions: Serum and urine might not serve as useful samples for the molecular diagnosis of cystic echinococcosis. However, PCR can be useful on serum samples to detect ruptured hydatid cysts and on hydatid cyst fluid to confirm the parasitic diagnosis. PMID:24754027

Performance of polymerase chain reaction for the diagnosis of cystic echinococcosis using serum, urine, and cyst fluid samples.

PubMed

Chaya, Dr; Parija, Subhash Chandra

2014-01-01

Cystic echinococcosis (CE) is a chronic zoonosis which presents with variable clinical manifestations. Currently the diagnosis of this disease is based on radiological findings and serological tests which lack specificity. Although antigen detection from the cyst fluid is the most specific, it is seldom done due to the complications involved. Detecting the presence of Echinococcus granulosus specific deoxyribonucleic acid (DNA) by the polymerase chain reaction (PCR) could provide a definitive diagnosis of CE. An in-house PCR assay was devised to detect E. granulosus specific DNA in serum, urine and hydatid cyst fluid. The ability of the PCR to detect E. granulosus in the above mentioned samples were observed in comparison with other antigen and antibody detection tests. Serum samples from surgically confirmed patients of CE with ruptured cysts contained the corresponding DNA while the in the majority of cases who had an intact cyst had no DNA of E. granulosus in their serum. DNA of E. granulosus was not found to be excreted in urine. PCR performed equal to antigen detection ELISA while testing hydatid cyst fluid samples. Serum and urine might not serve as useful samples for the molecular diagnosis of cystic echinococcosis. However, PCR can be useful on serum samples to detect ruptured hydatid cysts and on hydatid cyst fluid to confirm the parasitic diagnosis.

Diagnostic validation of three test methods for detection of cyprinid herpesvirus 3 (CyHV-3).

PubMed

Clouthier, Sharon C; McClure, Carol; Schroeder, Tamara; Desai, Megan; Hawley, Laura; Khatkar, Sunita; Lindsay, Melissa; Lowe, Geoff; Richard, Jon; Anderson, Eric D

2017-03-06

Cyprinid herpesvirus 3 (CyHV-3) is the aetiological agent of koi herpesvirus disease in koi and common carp. The disease is notifiable to the World Organisation for Animal Health. Three tests-quantitative polymerase chain reaction (qPCR), conventional PCR (cPCR) and virus isolation by cell culture (VI)-were validated to assess their fitness as diagnostic tools for detection of CyHV-3. Test performance metrics of diagnostic accuracy were sensitivity (DSe) and specificity (DSp). Repeatability and reproducibility were measured to assess diagnostic precision. Estimates of test accuracy, in the absence of a gold standard reference test, were generated using latent class models. Test samples originated from wild common carp naturally exposed to CyHV-3 or domesticated koi either virus free or experimentally infected with the virus. Three laboratories in Canada participated in the precision study. Moderate to high repeatability (81 to 99%) and reproducibility (72 to 97%) were observed for the qPCR and cPCR tests. The lack of agreement observed between some of the PCR test pair results was attributed to cross-contamination of samples with CyHV-3 nucleic acid. Accuracy estimates for the PCR tests were 99% for DSe and 93% for DSp. Poor precision was observed for the VI test (4 to 95%). Accuracy estimates for VI/qPCR were 90% for DSe and 88% for DSp. Collectively, the results show that the CyHV-3 qPCR test is a suitable tool for surveillance, presumptive diagnosis and certification of individuals or populations as CyHV-3 free.

Use of real-time PCR to detect canine parvovirus in feces of free-ranging wolves.

PubMed

Mech, L David; Almberg, Emily S; Smith, Douglas; Goyal, Sagar; Singer, Randall S

2012-04-01

Using real-time PCR, we tested 15 wolf (Canis lupus) feces from the Superior National Forest (SNF), Minnesota, USA, and 191 from Yellowstone National Park (YNP), USA, collected during summer and 13 during winter for canine parvovirus (CPV)-2 DNA. We also tested 20 dog feces for CPV-2 DNA. The PCR assay was 100% sensitive and specific with a minimum detection threshold of 10(4) 50% tissue culture infective dose. Virus was detected in two winter specimens but none of the summer specimens. We suggest applying the technique more broadly especially with winter feces.

Use of real-time PCR to detect canine parvovirus in feces of free-ranging wolves

USGS Publications Warehouse

Mech, L. David; Almberg, Emily S.; Smith, Douglas; Goyal, Sagar; Singer, Randall S.

2012-01-01

Using real-time PCR, we tested 15 wolf (Canis lupus) feces from the Superior National Forest (SNF), Minnesota, USA, and 191 from Yellowstone National Park (YNP), USA, collected during summer and 13 during winter for canine parvovirus (CPV)-2 DNA. We also tested 20 dog feces for CPV-2 DNA. The PCR assay was 100% sensitive and specific with a minimum detection threshold of 104 50% tissue culture infective dose. Virus was detected in two winter specimens but none of the summer specimens. We suggest applying the technique more broadly especially with winter feces.

Gold nanoparticle-based RT-PCR and real-time quantitative RT-PCR assays for detection of Japanese encephalitis virus

NASA Astrophysics Data System (ADS)

Huang, Su-Hua; Yang, Tsuey-Ching; Tsai, Ming-Hong; Tsai, I.-Shou; Lu, Huang-Chih; Chuang, Pei-Hsin; Wan, Lei; Lin, Ying-Ju; Lai, Chih-Ho; Lin, Cheng-Wen

2008-10-01

Virus isolation and antibody detection are routinely used for diagnosis of Japanese encephalitis virus (JEV) infection, but the low level of transient viremia in some JE patients makes JEV isolation from clinical and surveillance samples very difficult. We describe the use of gold nanoparticle-based RT-PCR and real-time quantitative RT-PCR assays for detection of JEV from its RNA genome. We tested the effect of gold nanoparticles on four different PCR systems, including conventional PCR, reverse-transcription PCR (RT-PCR), and SYBR green real-time PCR and RT-PCR assays for diagnosis in the acute phase of JEV infection. Gold nanoparticles increased the amplification yield of the PCR product and shortened the PCR time compared to the conventional reaction. In addition, nanogold-based real-time RT-PCR showed a linear relationship between Ct and template amount using ten-fold dilutions of JEV. The nanogold-based RT-PCR and real-time quantitative RT-PCR assays were able to detect low levels (1-10 000 copies) of the JEV RNA genomes extracted from culture medium or whole blood, providing early diagnostic tools for the detection of low-level viremia in the acute-phase infection. The assays described here were simple, sensitive, and rapid approaches for detection and quantitation of JEV in tissue cultured samples as well as clinical samples.

Electrochemical techniques on sequence-specific PCR amplicon detection for point-of-care applications.

PubMed

Luo, Xiaoteng; Hsing, I-Ming

2009-10-01

Nucleic acid based analysis provides accurate differentiation among closely affiliated species and this species- and sequence-specific detection technique would be particularly useful for point-of-care (POC) testing for prevention and early detection of highly infectious and damaging diseases. Electrochemical (EC) detection and polymerase chain reaction (PCR) are two indispensable steps, in our view, in a nucleic acid based point-of-care testing device as the former, in comparison with the fluorescence counterpart, provides inherent advantages of detection sensitivity, device miniaturization and operation simplicity, and the latter offers an effective way to boost the amount of targets to a detectable quantity. In this mini-review, we will highlight some of the interesting investigations using the combined EC detection and PCR amplification approaches for end-point detection and real-time monitoring. The promise of current approaches and the direction for future investigations will be discussed. It would be our view that the synergistic effect of the combined EC-PCR steps in a portable device provides a promising detection technology platform that will be ready for point-of-care applications in the near future.

Clinical and epidemiological use of nested PCR targeting the repetitive element IS1111 associated with the transposase gene from Coxiella burnetii.

PubMed

Mares-Guia, Maria Angélica M M; Guterres, Alexandro; Rozental, Tatiana; Ferreira, Michelle Dos Santos; Lemos, Elba R S

Q fever is a worldwide zoonosis caused by Coxiella burnetii-a small obligate intracellular Gram-negative bacterium found in a variety of animals. It is transmitted to humans by inhalation of contaminated aerosols from urine, feces, milk, amniotic fluid, placenta, abortion products, wool, and rarely by ingestion of raw milk from infected animals. Nested PCR can improve the sensitivity and specificity of testing while offering a suitable amplicon size for sequencing. Serial dilutions were performed tenfold to test the limit of detection, and the result was 10× detection of C. burnetti DNA with internal nested PCR primers relative to trans-PCR. Different biological samples were tested and identified only in nested PCR. This demonstrates the efficiency and effectiveness of the primers. Of the 19 samples, which amplify the partial sequence of C. burnetii, 12 were positive by conventional PCR and nested PCR. Seven samples-five spleen tissue samples from rodents and two tick samples-were only positive in nested PCR. With these new internal primers for trans-PCR, we demonstrate that our nested PCR assay for C. burnetii can achieve better results than conventional PCR. Published by Elsevier Editora Ltda.

Development of duplex RT-PCR-ELISA for the simultaneous detection of hepatitis A virus and hepatitis E virus.

PubMed

Tahk, Hongmin; Lee, Min Hwa; Lee, Kang Bum; Cheon, Doo-Sung; Choi, Changsun

2011-07-01

This study aimed to develop a specific and sensitive duplex reverse transcription polymerase chain reaction enzyme-linked immunosorbent assay (duplex RT-PCR-ELISA) for hepatitis A virus (HAV) and hepatitis E virus (HEV). Duplex RT-PCR-ELISA could detect and differentiate HAV and HEV with specific probes. When ELISA technique was used to detect probe-bound RT-PCR products, duplex RT-PCR-ELISA could detect as little as 0.1 ng/μL HAV and HEV from clinical samples. Human norovirus, enterovirus, poliovirus, murine norovirus and feline calicivirus were used for the specificity test; all were negative. Therefore duplex RT-PCR-ELISA can be used for the simultaneous detection of HAV and HEV in contaminated fecal samples. Copyright © 2011 Elsevier B.V. All rights reserved.

Novel approach based on one-tube nested PCR and a lateral flow strip for highly sensitive diagnosis of tuberculous meningitis.

PubMed

Sun, Yajuan; Chen, Jiajun; Li, Jia; Xu, Yawei; Jin, Hui; Xu, Na; Yin, Rui; Hu, Guohua

2017-01-01

Rapid and sensitive detection of Mycobacterium tuberculosis (M. Tb) in cerebrospinal fluid is crucial in the diagnosis of tuberculous meningitis (TBM), but conventional diagnostic technologies have limited sensitivity and specificity or are time-consuming. In this work, a novel, highly sensitive molecular diagnostic method, one-tube nested PCR-lateral flow strip test (OTNPCR-LFST), was developed for detecting M. tuberculosis. This one-tube nested PCR maintains the sensitivity of conventional two-step nested PCR and reduces both the chance of cross-contamination and the time required for analysis. The PCR product was detected by a lateral flow strip assay, which provided a basis for migration of the test to a point-of-care (POC) microfluidic format. The developed assay had an improved sensitivity compared with traditional PCR, and the limit of detection was up to 1 fg DNA isolated from M. tuberculosis. The assay was also specific for M. tuberculosis, and no cross-reactions were found in other non-target bacteria. The application of this technique to clinical samples was successfully evaluated, and OTNPCR-LFST showed 89% overall sensitivity and 100% specificity for TBM patients. This one-tube nested PCR-lateral flow strip assay is useful for detecting M. tuberculosis in TBM due to its rapidity, high sensitivity and simple manipulation.

Development and evaluation of a quantitative PCR assay for detection of Hepatozoon sp.

PubMed

Criado-Fornelio, A; Buling, A; Cunha-Filho, N A; Ruas, J L; Farias, N A R; Rey-Valeiron, C; Pingret, J L; Etievant, M; Barba-Carretero, J C

2007-12-25

With the aim to improve current molecular diagnostic techniques of Hepatozoon sp. in carnivore mammals, we developed a quantitative PCR (qPCR) assay with SYBR Green I((R)). The method, consisting of amplification of a 235bp fragment of the 18S rRNA gene, is able to detect at least 0.1fg of parasite DNA. Reproducible quantitative results were obtained over a range of 0.1ng-0.1fg of Hepatozoon sp. DNA. To assess the performance of the qPCR assay, DNA samples from dogs (140) and cats (50) were tested with either standard PCR or qPCR. Positive samples were always confirmed by partial sequencing of the 18S rRNA gene. Quantitative PCR was 15.8% more sensitive than standard PCR to detect H. canis in dogs. In cats, no infections were detected by standard PCR, compared to two positives by qPCR (which were infected by H. canis as shown by sequencing).

Comparison of culture, single and multiplex real-time PCR for detection of Sabin poliovirus shedding in recently vaccinated Indian children.

PubMed

Giri, Sidhartha; Rajan, Anand K; Kumar, Nirmal; Dhanapal, Pavithra; Venkatesan, Jayalakshmi; Iturriza-Gomara, Miren; Taniuchi, Mami; John, Jacob; Abraham, Asha Mary; Kang, Gagandeep

2017-08-01

Although, culture is considered the gold standard for poliovirus detection from stool samples, real-time PCR has emerged as a faster and more sensitive alternative. Detection of poliovirus from the stool of recently vaccinated children by culture, single and multiplex real-time PCR was compared. Of the 80 samples tested, 55 (68.75%) were positive by culture compared to 61 (76.25%) and 60 (75%) samples by the single and one step multiplex real-time PCR assays respectively. Real-time PCR (singleplex and multiplex) is more sensitive than culture for poliovirus detection in stool, although the difference was not statistically significant. © 2017 Wiley Periodicals, Inc.

Microwell hybridization assay for detection of PCR products from Mycobacterium tuberculosis complex and the recombinant Mycobacterium smegmatis strain 1008 used as an internal control.

PubMed Central

Kox, L F; Noordhoek, G T; Kunakorn, M; Mulder, S; Sterrenburg, M; Kolk, A H

1996-01-01

A microwell hybridization assay was developed for the detection of the PCR products from both Mycobacterium tuberculosis complex bacteria and the recombinant Mycobacterium smegmatis strain 1008 that is used as an internal control to monitor inhibition in the PCR based on the M. tuberculosis complex-specific insertion sequence IS6110. The test is based on specific detection with digoxigenin-labeled oligonucleotide probes of biotinylated PCR products which are captured in a microtiter plate coated with streptavidin. The captured PCR products are hybridized separately with two probes, one specific for the PCR product from IS6110 from M. tuberculosis complex and the other specific for the PCR fragment from the modified IS6110 fragment from the recombinant M. smegmatis 1008. The microwell hybridization assay discriminates perfectly between the two types of amplicon. The amount of PCR product that can be detected by this assay is 10 times less than that which can be detected by agarose gel electrophoresis. The test can be performed in 2 h. It is much faster and less laborious than Southern blot hybridization. Furthermore, the interpretation of results is objective. The assay was used with 172 clinical samples in a routine microbiology laboratory, and the results were in complete agreement with those of agarose gel electrophoresis and Southern blot hybridization. PMID:8862568

A novel RT-PCR for the detection of Helicobacter pylori and identification of clarithromycin resistance mediated by mutations in the 23S rRNA gene.

PubMed

Redondo, Javier Jareño; Keller, Peter M; Zbinden, Reinhard; Wagner, Karoline

2018-01-01

In this study we evaluated the commercially available LightMix® RT-PCR assay for Helicobacter pylori detection and identification of clarithromycin (CLR) resistance in culture and clinical specimens (gastric biopsies and stool). The H. pylori LightMix® RT-PCR detects a 97bp long fragment of the 23S rRNA gene and allows the identification of 3 distinct point mutations conferring CLR resistance via melting curve analysis. The performance of the H. pylori LightMix® RT-PCR was evaluated using a set of 60 H. pylori strains showing phenotypical CLR susceptibility or CLR resistance (Minimum inhibitory concentrations from 0.016 to 256mg/L). We found high concordance (95%) between phenotypical CLR resistance screening by E-Test® and the Lightmix® RT-PCR. Discrepant results were verified by sequencing of the 23S rRNA gene that always confirmed the results obtained by Lightmix® RT-PCR. Furthermore, H. pylori was detected in clinical biopsy and stool specimens by Lightmix® RT-PCR that identified the correct H. pylori genotype. The LightMix® RT-PCR is an accurate, sensitive and easy to use test for H. pylori and CLR resistance detection and can therefore be readily implemented in any diagnostic laboratory. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

PCR-Based Method for Detecting Viral Penetration of Medical Exam Gloves

PubMed Central

Broyles, John M.; O'Connell, Kevin P.; Korniewicz, Denise M.

2002-01-01

The test approved by the U.S. Food and Drug Administration for assessment of the barrier quality of medical exam gloves includes visual inspection and a water leak test. Neither method tests directly the ability of gloves to prevent penetration by microorganisms. Methods that use microorganisms (viruses and bacteria) to test gloves have been developed but require classical culturing of the organism to detect it. We have developed a PCR assay for bacteriophage φX174 that allows the rapid detection of penetration of gloves by this virus. The method is suitable for use with both latex and synthetic gloves. The presence of glove powder on either latex or synthetic gloves had no effect on the ability of the PCR assay to detect bacteriophage DNA. The assay is rapid, sensitive, and inexpensive; requires only small sample volumes; and can be automated. PMID:12149320

Stem loop-mediated isothermal amplification test: comparative analysis with classical LAMP and PCR in detection of Entamoeba histolytica in Kenya.

PubMed

Mwendwa, Fridah; Mbae, Cecilia K; Kinyua, Johnson; Mulinge, Erastus; Mburugu, Gitonga Nkanata; Njiru, Zablon K

2017-03-31

Entamoeba histolytica, the causative agent for amoebiasis is a considerable burden to population in the developing countries where it accounts for over 50 million infections. The tools for detection of amoebiasis are inadequate and diagnosis relies on microscopy which means a significant percent of cases remain undiagnosed. Moreover, tests formats that can be rapidly applied in rural endemic areas are not available. In this study, a loop-mediated isothermal test (LAMP) based on 18S small subunit ribosomal RNA gene was designed with extra reaction accelerating primers (stem primers) and compared with the published LAMP and PCR tests in detection of E. histolytica DNA in clinical samples. The stem LAMP test indicated shorter time to results by an average 11 min and analytical sensitivity of 10 -7 (~30 pg/ml) compared to the standard LAMP and PCR which showed sensitivities levels of 10 -5 (~3 ng/ml) and 10 -4 (~30 ng/ml) respectively using tenfold serial dilution of DNA. In the analysis of clinical specimens positive for Entamoeba spp. trophozoites and cysts using microscopy, the stem LAMP test detected E. histolytica DNA in 36/126, standard LAMP test 20/126 and PCR 17/126 cases respectively. There was 100% agreement in detection of the stem LAMP test product using fluorescence of SYTO-9 dye in real time machine, through addition of 1/10 dilution of SYBR ® Green I and electrophoresis in 2% agarose gel stained with ethidium bromide. The stem LAMP test developed in this study indicates potential towards detection of E. histolytica.

Detection of coliform bacteria and Escherichia coli by multiplex polymerase chain reaction: comparison with defined substrate and plating methods for water quality monitoring.

PubMed Central

Bej, A K; McCarty, S C; Atlas, R M

1991-01-01

Multiplex polymerase chain reaction (PCR) and gene probe detection of target lacZ and uidA genes were used to detect total coliform bacteria and Escherichia coli, respectively, for determining water quality. In tests of environmental water samples, the lacZ PCR method gave results statistically equivalent to those of the plate count and defined substrate methods accepted by the U.S. Environmental Protection Agency for water quality monitoring and the uidA PCR method was more sensitive than 4-methylumbelliferyl-beta-D-glucuronide-based defined substrate tests for specific detection of E. coli. Images PMID:1768116

Performance of automated multiplex PCR using sonication fluid for diagnosis of periprosthetic joint infection: a prospective cohort.

PubMed

Renz, Nora; Feihl, Susanne; Cabric, Sabrina; Trampuz, Andrej

2017-12-01

Sonication of explanted prostheses improved the microbiological diagnosis of periprosthetic joint infections (PJI). We evaluated the performance of automated multiplex polymerase chain reaction (PCR) using sonication fluid for the microbiological diagnosis of PJI. In a prospective cohort using uniform definition criteria for PJI, explanted joint prostheses were investigated by sonication and the resulting sonication fluid was analyzed by culture and multiplex PCR. McNemar's Chi-squared test was used to compare the performance of diagnostic tests. Among 111 patients, PJI was diagnosed in 78 (70%) and aseptic failure in 33 (30%). For the diagnosis of PJI, the sensitivity and specificity of periprosthetic tissue culture was 51 and 100%, of sonication fluid culture 58 and 100%, and of sonication fluid PCR 51 and 94%, respectively. Among 70 microorganisms, periprosthetic tissue culture grew 52 (74%), sonication fluid culture grew 50 (71%) and sonication fluid PCR detected 37 pathogens (53%). If only organisms are considered, for which primers are included in the test panel, PCR detected 37 of 58 pathogens (64%). The sonication fluid PCR missed 19 pathogens (predominantly oral streptococci and anaerobes), whereas 7 additional microorganisms were detected only by PCR (including Cutibacterium spp. and coagulase-negative staphylococci). The performance of multiplex PCR using sonication fluid is comparable to culture of periprosthetic tissue or sonication fluid. The advantages of PCR are short processing time (< 5 h) and fully automated procedure. However, culture technique is still needed due to the low sensitivity and the need of comprehensive susceptibility testing. Modification of primers or inclusion of additional ones may improve the performance of PCR, especially of low-virulent organisms.

MULTIPLEX SYBR® GREEN-REAL TIME PCR (qPCR) ASSAY FOR THE DETECTION AND DIFFERENTIATION OF Bartonella henselae AND Bartonella clarridgeiae IN CATS

PubMed Central

Staggemeier, Rodrigo; Pilger, Diogo André; Spilki, Fernando Rosado; Cantarelli, Vlademir Vicente

2014-01-01

A novel SYBR® green-real time polymerase chain reaction (qPCR) was developed to detect two Bartonella species, B. henselae and B. clarridgeiae, directly from blood samples. The test was used in blood samples obtained from cats living in animal shelters in Southern Brazil. Results were compared with those obtained by conventional PCR targeting Bartonella spp. Among the 47 samples analyzed, eight were positive using the conventional PCR and 12 were positive using qPCR. Importantly, the new qPCR detected the presence of both B. henselae and B. clarridgeiae in two samples. The results show that the qPCR described here may be a reliable tool for the screening and differentiation of two important Bartonella species. PMID:24626408

Multiplex SYBR® green-real time PCR (qPCR) assay for the detection and differentiation of Bartonella henselae and Bartonella clarridgeiae in cats.

PubMed

Staggemeier, Rodrigo; Pilger, Diogo André; Spilki, Fernando Rosado; Cantarelli, Vlademir Vicente

2014-01-01

A novel SYBR® green-real time polymerase chain reaction (qPCR) was developed to detect two Bartonella species, B. henselae and B. clarridgeiae, directly from blood samples. The test was used in blood samples obtained from cats living in animal shelters in Southern Brazil. Results were compared with those obtained by conventional PCR targeting Bartonella spp. Among the 47 samples analyzed, eight were positive using the conventional PCR and 12 were positive using qPCR. Importantly, the new qPCR detected the presence of both B. henselae and B. clarridgeiae in two samples. The results show that the qPCR described here may be a reliable tool for the screening and differentiation of two important Bartonella species.

Real-Time PCR with an Internal Control for Detection of All Known Human Adenovirus Serotypes▿

PubMed Central

Damen, Marjolein; Minnaar, René; Glasius, Patricia; van der Ham, Alwin; Koen, Gerrit; Wertheim, Pauline; Beld, Marcel

2008-01-01

The “gold standard” for the diagnosis of adenovirus (AV) infection is virus culture, which is rather time-consuming. Especially for immunocompromised patients, in whom severe infections with AV have been described, rapid diagnosis is important. Therefore, an internally controlled AV real-time PCR assay detecting all known human AV serotypes was developed. Primers were chosen from the hexon region, which is the most conserved region, and in order to cover all known serotypes, degenerate primers were used. The internal control (IC) DNA contained the same primer binding sites as the AV DNA control but had a shuffled probe region compared to the conserved 24-nucleotide consensus AV hexon probe region (the target). The IC DNA was added to the clinical sample in order to monitor extraction and PCR efficiency. The sensitivity and the linearity of the AV PCR were determined. For testing the specificity of this PCR assay for human AVs, a selection of 51 AV prototype strains and 66 patient samples positive for other DNA viruses were tested. Moreover, a comparison of the AV PCR method described herein with culture and antigen (Ag) detection was performed with a selection of 151 clinical samples. All 51 AV serotypes were detected in the selection of AV prototype strains. Concordant results from culture or Ag detection and PCR were found for 139 (92.1%) of 151 samples. In 12 cases (7.9%), PCR was positive while the culture was negative. In conclusion, a sensitive, internally controlled nonnested AV real-time PCR assay which is able to detect all known AV serotypes with higher sensitivity than a culture or Ag detection method was developed. PMID:18923006

Human papillomavirus detection using the Abbott RealTime high-risk HPV tests compared with conventional nested PCR coupled to high-throughput sequencing of amplification products in cervical smear specimens from a Gabonese female population.

PubMed

Moussavou-Boundzanga, Pamela; Koumakpayi, Ismaël Hervé; Labouba, Ingrid; Leroy, Eric M; Belembaogo, Ernest; Berthet, Nicolas

2017-12-21

Cervical cancer is the fourth most common malignancy in women worldwide. However, screening with human papillomavirus (HPV) molecular tests holds promise for reducing cervical cancer incidence and mortality in low- and middle-income countries. The performance of the Abbott RealTime High-Risk HPV test (AbRT) was evaluated in 83 cervical smear specimens and compared with a conventional nested PCR coupled to high-throughput sequencing (HTS) to identify the amplicons. The AbRT assay detected at least one HPV genotype in 44.57% of women regardless of the grade of cervical abnormalities. Except for one case, good concordance was observed for the genotypes detected with the AbRT assay in the high-risk HPV category determined with HTS of the amplicon generated by conventional nested PCR. The AbRT test is an easy and reliable molecular tool and was as sensitive as conventional nested PCR in cervical smear specimens for detection HPVs associated with high-grade lesions. Moreover, sequencing amplicons using an HTS approach effectively identified the genotype of the hrHPV identified with the AbRT test.

Development and validation of a duplex real-time PCR assay for the diagnosis of equine piroplasmosis.

PubMed

Lobanov, Vladislav A; Peckle, Maristela; Massard, Carlos L; Brad Scandrett, W; Gajadhar, Alvin A

2018-03-02

Equine piroplasmosis (EP) is an economically significant infection of horses and other equine species caused by the tick-borne protozoa Theileria equi and Babesia caballi. The long-term carrier state in infected animals makes importation of such subclinical cases a major risk factor for the introduction of EP into non-enzootic areas. Regulatory testing for EP relies on screening of equines by serological methods. The definitive diagnosis of EP infection in individual animals will benefit from the availability of sensitive direct detection methods, for example, when used as confirmatory assays for non-negative serological test results. The objectives of this study were to develop a real-time quantitative polymerase chain reaction (qPCR) assay for simultaneous detection of both agents of EP, perform comprehensive evaluation of its performance and assess the assay's utility for regulatory testing. We developed a duplex qPCR targeting the ema-1 gene of T. equi and the 18S rRNA gene of B. caballi and demonstrated that the assay has high analytical sensitivities for both piroplasm species. Validation of the duplex qPCR on samples from 362 competitive enzyme-linked immunosorbent assay (cELISA)-negative horses from Canada and the United States yielded no false-positive reactions. The assay's performance was further evaluated using samples collected from 430 horses of unknown EP status from a highly endemic area in Brazil. This set of samples was also tested by a single-target 18S rRNA qPCR for T. equi developed at the OIE reference laboratory for EP in Japan, and a previously published single-target 18S rRNA qPCR for B. caballi whose oligonucleotides we adopted for use in the duplex qPCR. Matching serum samples were tested for antibodies to these parasites using cELISA. By the duplex qPCR, T. equi-specific 18S rRNA qPCR and cELISA, infections with T. equi were detected in 87.9% (95% confidence interval, CI: 84.5-90.7%), 90.5% (95% CI: 87.3-92.3%) and 87.4% (95% CI: 84.0-90.2%) of the horses, respectively. The B. caballi prevalence estimates were 9.3% (95% CI: 6.9-12.4%) by the duplex qPCR and 7.9% (95% CI: 5.7-10.9%) by the respective single-target qPCR assay. These values were markedly lower compared to the seroprevalence of 58.6% (95% CI: 53.9-63.2%) obtained by B. caballi-specific cELISA. The relative diagnostic sensitivity of the duplex qPCR for T. equi was 95.5%, as 359 of the 376 horses with exposure to T. equi confirmed by cELISA had parasitemia levels above the detection limit of the molecular assay. In contrast, only 39 (15.5%) of the 252 horses with detectable B. caballi-specific antibodies were positive for this piroplasm species by the duplex qPCR. The duplex qPCR described here performed comparably to the existing single-target qPCR assays for T. equi and B. caballi and will be more cost-effective in terms of results turnaround time and reagent costs when both pathogens are being targeted for disease control and epidemiological investigations. These validation data also support the reliability of the ema-1 gene-specific oligonucleotides developed in this study for confirmatory testing of non-negative serological test results for T. equi by qPCR. However, the B. caballi-specific qPCR cannot be similarly recommended as a confirmatory assay for routine regulatory testing due to the low level of agreement with serological test results demonstrated in this study. Further studies are needed to determine the transmission risk posed by PCR-negative equines with detectable antibodies to B. caballi.

African Swine Fever Diagnosis Adapted to Tropical Conditions by the Use of Dried-blood Filter Papers.

PubMed

Randriamparany, T; Kouakou, K V; Michaud, V; Fernández-Pinero, J; Gallardo, C; Le Potier, M-F; Rabenarivahiny, R; Couacy-Hymann, E; Raherimandimby, M; Albina, E

2016-08-01

The performance of Whatman 3-MM filter papers for the collection, drying, shipment and long-term storage of blood at ambient temperature, and for the detection of African swine fever virus and antibodies was assessed. Conventional and real-time PCR, viral isolation and antibody detection by ELISA were performed on paired samples (blood/tissue versus dried-blood 3-MM filter papers) collected from experimentally infected pigs and from farm pigs in Madagascar and Côte d'Ivoire. 3-MM filter papers were used directly in the conventional and real-time PCR without previous extraction of nucleic acids. Tests that performed better with 3-MM filter papers were in descending order: virus isolation, real-time UPL PCR and conventional PCR. The analytical sensitivity of real-time UPL PCR on filter papers was similar to conventional testing (virus isolation or conventional PCR) on organs or blood. In addition, blood-dried filter papers were tested in ELISA for antibody detection and the observed sensitivity was very close to conventional detection on serum samples and gave comparable results. Filter papers were stored up to 9 months at 20-25°C and for 2 months at 37°C without significant loss of sensitivity for virus genome detection. All tests on 3-MM filter papers had 100% specificity compared to the gold standards. Whatman 3-MM filter papers have the advantage of being cheap and of preserving virus viability for future virus isolation and characterization. In this study, Whatman 3-MM filter papers proved to be a suitable support for the collection, storage and use of blood in remote areas of tropical countries without the need for a cold chain and thus provide new possibilities for antibody testing and virus isolation. © 2014 Blackwell Verlag GmbH.

Development of a multiplex PCR assay for detection and discrimination of Theileria annulata and Theileria sergenti in cattle.

PubMed

Junlong, Liu; Li, Youquan; Liu, Aihong; Guan, Guiquan; Xie, Junren; Yin, Hong; Luo, Jianxun

2015-07-01

Aim to construct a simple and efficient diagnostic assay for Theileria annulata and Theileria sergenti, a multiplex polymerase chain reaction (PCR) method was developed in this study. Following the alignment of the related sequences, two primer sets were designed specific targeting on T. annulata cytochrome b (COB) gene and T. sergenti internal transcribed spacer (ITS) sequences. It was found that the designed primers could react in one PCR system and generating amplifications of 818 and 393 base pair for T. sergenti and T. annulata, respectively. The standard genomic DNA of both species Theileria was serial tenfold diluted for testing the sensitivity, while specificity test confirmed both primer sets have no cross-reaction with other Theileria and Babesia species. In addition, 378 field samples were used for evaluation of the utility of the multiplex PCR assay for detection of the pathogens infection. The detection results were compared with the other two published PCR methods which targeting on T. annulata COB gene and T. sergenti major piroplasm surface protein (MPSP) gene, respectively. The developed multiplex PCR assay has similar efficient detection with COB and MPSP PCR, which indicates this multiplex PCR may be a valuable assay for the epidemiological studies for T. annulata and T. sergenti.

Enterovirus D68 detection in respiratory specimens: Association with severe disease.

PubMed

Engelmann, Ilka; Fatoux, Marie; Lazrek, Mouna; Alidjinou, Enagnon K; Mirand, Audrey; Henquell, Cécile; Dewilde, Anny; Hober, Didier

2017-07-01

Molecular techniques increased the number of documented respiratory infections. In a substantial number of cases the causative agent remains undetected. Since August 2014, an increase in Enterovirus(EV)-D68 infections was reported. We aimed to investigate epidemiology and clinical relevance of EV-D68. From June to December 2014 and from September to December 2015, 803 and 847 respiratory specimens, respectively, were tested for respiratory viruses with a multiplex RT-PCR. This multiplex RT-PCR does not detect EV-D68. Therefore, 457 (2014) and 343 (2015) specimens with negative results were submitted to an EV-specific-RT-PCR. EV-positive specimens were tested with an EV-D68-specific-RT-PCR and genotyped. Eleven specimens of 2014 tested positive in the EV-specific-RT-PCR and of these seven were positive in the EV-D68-specific-RT-PCR. Typing confirmed these as EV-D68. Median age of EV-D68-positive patients was 3 years (1 month-91 years). Common symptoms included fever (n = 6, 86%), respiratory distress (n = 5, 71%), and cough (n = 4, 57%). All EV-D68-positive patients were admitted to hospital, 4 (57%) were admitted to intensive care units and 6 (86%) received oxygen. One patient suffered from acute flaccid paralysis. Seven specimens of 2015 were positive in the EV-specific-RT-PCR but negative in the EV-D68-specific-RT-PCR. In conclusion, use of an EV-specific-RT-PCR allowed us to detect EV-D68 circulation in autumn 2014 that was not detected by the multiplex RT-PCR and was associated with severe disease. © 2017 Wiley Periodicals, Inc.

High-resolution melting PCR assay, applicable for diagnostics and screening studies, allowing detection and differentiation of several Babesia spp. infecting humans and animals.

PubMed

Rozej-Bielicka, Wioletta; Masny, Aleksander; Golab, Elzbieta

2017-10-01

The goal of the study was to design a single tube PCR test for detection and differentiation of Babesia species in DNA samples obtained from diverse biological materials. A multiplex, single tube PCR test was designed for amplification of approximately 400 bp region of the Babesia 18S rRNA gene. Universal primers were designed to match DNA of multiple Babesia spp. and to have low levels of similarity to DNA sequences of other intracellular protozoa and Babesia hosts. The PCR products amplified from Babesia DNA isolated from human, dog, rodent, deer, and tick samples were subjected to high-resolution melting analysis for Babesia species identification. The designed test allowed detection and differentiation of four Babesia species, three zoonotic (B. microti, B. divergens, B. venatorum) and one that is generally not considered zoonotic-Babesia canis. Both detection and identification of all four species were possible based on the HRM curves of the PCR products in samples obtained from the following: humans, dogs, rodents, and ticks. No cross-reactivity with DNA of Babesia hosts or Plasmodium falciparum and Toxoplasma gondii was observed. The lack of cross-reactivity with P. falciparum DNA might allow using the assay in endemic malaria areas. The designed assay is the first PCR-based test for detection and differentiation of several Babesia spp. of medical and veterinary importance, in a single tube reaction. The results of the study show that the designed assay for Babesia detection and identification could be a practical and inexpensive tool for diagnostics and screening studies of diverse biological materials.

Evolution of Specific Antibodies and Proviral DNA in Milk of Small Ruminants Infected by Small Ruminant Lentivirus

PubMed Central

Barquero, Nuria; Gomez-Lucia, Esperanza; Arjona, Alvaro; Toural, Cristina; las Heras, Alfonso; Fernández-Garayzabal, José F.; Domenech, Ana

2013-01-01

The diagnosis of Small Ruminant Lentivirus (SRLV) is based on clinical signs, pathological lesions and laboratory testing. No standard reference test for the diagnosis of maedi visna has been validated up to the present, and it is puzzling that tests which detect antibodies against the virus and tests which detect the proviral genome may render opposite results. The aim of this study was to evaluate the presence in milk throughout a lactation period of specific antibodies by ELISA and of SRLV proviral DNA by a PCR of the highly conserved pol region. A six-month study was conducted with the milk of 28 ewes and 31 goats intensively reared. The percentage of animals with antibodies against SRLV increased throughout the study period. Seroprevalence in sheep was 28% at the beginning of the study and by the end it had increased up to 52.4%. In goats, initial seroprevalence of 5.6% increased to 16%. The percentage of PCR positive ewes was stable throughout the study period. Of the positive sheep, 21.4% were PCR-positive before antibodies could be detected and most of them became PCR-negative shortly after the first detection of antibodies. This might suggest that antibodies have a neutralizing effect. In addition, an equal percentage of sheep were always PCR-negative but either became ELISA-positive or was always ELISA-positive, which might support this hypothesis. On the other hand, the PCR results in goats did not follow any pattern and oscillated between 35.3% and 55.6% depending on the month. Most goats positive by PCR failed to develop antibodies in the 6 months tested. We may conclude that the infection and the antibody response to it follow a different trend in sheep and goats. PMID:24153063

A serotype-specific polymerase chain reaction for identification of Pasteurella multocida serotype 1

USGS Publications Warehouse

Rocke, T.E.; Smith, S.R.; Miyamoto, A.; Shadduck, D.J.

2002-01-01

A serotype-specific polymerase chain reaction (PCR) assay was developed for detection and identification of Pasteurella multocida serotype 1, the causative agent of avian cholera in wild waterfowl. Arbitrarily primed PCR was used to detect DNA fragments that distinguish serotype 1 from the other 15 serotypes of P. multocida (with the exception of serotype 14). Oligonucleotide primers were constructed from these sequences, and a PCR assay was optimized and evaluated. PCR reactions consistently resulted in amplification products with reference strains 1 and 14 and all other serotype 1 strains tested, with cell numbers as low as 2.3 cells/ml. No amplification products were produced with other P. multocida serotypes or any other bacterial species tested. To compare the sensitivity and further test the specificity of this PCR assay with traditional culturing and serotyping techniques, tissue samples from 84 Pekin ducks inoculated with field strains of P. multocida and 54 wild lesser snow geese collected during an avian cholera outbreak were provided by other investigators working on avian cholera. PCR was as sensitive (58/64) as routine isolation (52/64) in detecting and identifying P. multocida serotype 1 from the livers of inoculated Pekins that became sick or died from avian cholera. No product was amplified from tissues of 20 other Pekin ducks that received serotypes other than type 1 (serotype 3, 12 × 3, or 10) or 12 control birds. Of the 54 snow geese necropsied and tested for P. multocida, our PCR detected and identified the bacteria from 44 compared with 45 by direct isolation. The serotype-specific PCR we developed was much faster and less labor intensive than traditional culturing and serotyping procedures and could result in diagnosis of serotype 1 pasteurellosis within 24 hr of specimen submission.

Determining the analytical specificity of PCR-based assays for the diagnosis of IA: What is Aspergillus?

PubMed

Morton, C Oliver; White, P Lewis; Barnes, Rosemary A; Klingspor, Lena; Cuenca-Estrella, Manuel; Lagrou, Katrien; Bretagne, Stéphane; Melchers, Willem; Mengoli, Carlo; Caliendo, Angela M; Cogliati, Massimo; Debets-Ossenkopp, Yvette; Gorton, Rebecca; Hagen, Ferry; Halliday, Catriona; Hamal, Petr; Harvey-Wood, Kathleen; Jaton, Katia; Johnson, Gemma; Kidd, Sarah; Lengerova, Martina; Lass-Florl, Cornelia; Linton, Chris; Millon, Laurence; Morrissey, C Orla; Paholcsek, Melinda; Talento, Alida Fe; Ruhnke, Markus; Willinger, Birgit; Donnelly, J Peter; Loeffler, Juergen

2017-06-01

A wide array of PCR tests has been developed to aid the diagnosis of invasive aspergillosis (IA), providing technical diversity but limiting standardisation and acceptance. Methodological recommendations for testing blood samples using PCR exist, based on achieving optimal assay sensitivity to help exclude IA. Conversely, when testing more invasive samples (BAL, biopsy, CSF) emphasis is placed on confirming disease, so analytical specificity is paramount. This multicenter study examined the analytical specificity of PCR methods for detecting IA by blind testing a panel of DNA extracted from a various fungal species to explore the range of Aspergillus species that could be detected, but also potential cross reactivity with other fungal species. Positivity rates were calculated and regression analysis was performed to determine any associations between technical specifications and performance. The accuracy of Aspergillus genus specific assays was 71.8%, significantly greater (P < .0001) than assays specific for individual Aspergillus species (47.2%). For genus specific assays the most often missed species were A. lentulus (25.0%), A. versicolor (24.1%), A. terreus (16.1%), A. flavus (15.2%), A. niger (13.4%), and A. fumigatus (6.2%). There was a significant positive association between accuracy and using an Aspergillus genus PCR assay targeting the rRNA genes (P = .0011). Conversely, there was a significant association between rRNA PCR targets and false positivity (P = .0032). To conclude current Aspergillus PCR assays are better suited for detecting A. fumigatus, with inferior detection of most other Aspergillus species. The use of an Aspergillus genus specific PCR assay targeting the rRNA genes is preferential. © The Author 2016. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Multiple outbreaks of Norwalk-like virus gastro-enteritis associated with a Mediterranean-style restaurant.

PubMed

Marshall, J A; Yuen, L K; Catton, M G; Gunesekere, I C; Wright, P J; Bettelheim, K A; Griffith, J M; Lightfoot, D; Hogg, G G; Gregory, J; Wilby, R; Gaston, J

2001-02-01

The role of diverse infectious agents, particularly Norwalk-like viruses (NLV), in three successive gastro-enteritis outbreaks in one setting (a restaurant) was evaluated. Methods included standard bacteriological tests, specific tests for Escherichia coli, tests for verocytotoxins, electron microscopy (EM) for viruses and reverse transcription-PCR (RT-PCR) methodology for NLV. No pathogenic bacteria were detected. Verocytotoxin genes, although detected by PCR in the first outbreak, could not be confirmed in the E. coli isolated, so they did not appear to be of significance. NLV was the main agent detected in each of the three outbreaks. DNA sequencing and phylogenetic analysis of the amplified products obtained from the RT-PCR positive specimens indicated that only one NLV strain was involved in each outbreak, but the NLV strains responsible for the three outbreaks were different from each other. PCR technology for detection of NLV proved highly sensitive, but failed to detect one specimen which was positive by EM. The restaurant associated with the outbreaks is a Mediterranean-style restaurant where food from a common platter is typically eaten with fingers. The findings indicate that NLV was introduced by guests or staff and was not due to a long-term reservoir within the setting.

Comparative evaluation of the nested ITS PCR against the 18S PCR-RFLP in a survey of bovine trypanosomiasis in Kwale County, Kenya.

PubMed

Odongo, Steven; Delespaux, Vincent; Ngotho, Maina; Bekkele, Serkalem Mindaye; Magez, Stefan

2016-09-01

We compared the nested internal transcribed spacer (ITS) PCR and the 18S PCR-RFLP (restriction-fragment length polymorphism) pan-trypanosome assays in a cross-sectional survey of bovine trypanosomiasis in 358 cattle in Kwale County, Kenya. The prevalence of trypanosomiasis as determined by the nested ITS PCR was 19.6% (70/358) and by 18S PCR-RFLP was 16.8% (60/358). Of the pathogenic trypanosomes detected, the prevalence of Trypanosoma congolense and Trypanosoma vivax was greater than that of Trypanosoma simiae The nested ITS PCR detected 83 parasite events, whereas the 18S PCR-RFLP detected 64; however, overall frequencies of infections and the parasite events detected did not differ between the assays (χ(2) = 0.8, df = 1, p > 0.05 and χ(2) = 2.5, df = 1, p > 0.05, respectively). The kappa statistic (0.8) showed good agreement between the tests. The nested ITS PCR and the 18S PCR-RFLP had comparable sensitivity, although the nested ITS PCR was better at detecting mixed infections (χ(2) = 5.4, df = 1, p < 0.05). © 2016 The Author(s).

A multiplex PCR method for detection of Aspergillus spp. and Mycobacterium tuberculosis in BAL specimens.

PubMed

Amini, F; Kachuei, R; Noorbakhsh, F; Imani Fooladi, A A

2015-06-01

The aim of this study was the detection of Aspergillus species and Mycobacterium tuberculosis together in bronchoalveolar lavage (BAL) using of multiplex PCR. In this study, from September 2012 until June 2013, 100 bronchoalveolar lavage (BAL) specimens were collected from patients suspected of tuberculosis (TB). After the direct and culture test, multiplex PCR were utilized in order to diagnose Aspergillus species and M. tuberculosis. Phenol-chloroform manual method was used in order to extract DNA from these microorganisms. Aspergillus specific primers, M. tuberculosis designed primers and beta actin primers were used for multiplex PCR. In this study, by multiplex PCR method, Aspergillus species were identified in 12 samples (12%), positive samples in direct and culture test were respectively 11% and 10%. Sensitivity and specificity of this method in comparison to direct test were respectively 100% and 98.8%, also sensitivity and specificity of this method in comparison to culture test were respectively 100% and 97.7%. In this assay, M. tuberculosis was identified in 8 samples (8%). Mycobacterium-positive samples in molecular method, direct and culture test were respectively 6%, 5% and 7%. Sensitivity and specificity of PCR method in comparison to direct test were 80% and 97.8% also sensitivity and specificity of this method in comparison to culture test was 71.4% and 98.9%. In the present study, multiplex PCR method had higher sensitivity than direct and culture test in order to identify and detect Aspergillus, also this method had lower sensitivity for identification of M. tuberculosis, suggesting that the method of DNA extraction was not suitable. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

[Contribution of nested PCR in the diagnosis of imported malaria in southern Algeria].

PubMed

Bouiba, L; Gassen, B; Gasmi, M; Hammadi, D; Harrat, Z

2016-12-01

The nested PCR was used to estimate its inputs in malaria diagnosis and in the performance of the microscope operators involved in the surveillance of malaria in remote areas of South Algeria. For the period 2010 to 2015, 112 patients (93 febrile and 19 asymptomatic) coming from sub-Saharan Africa were tested for malaria in the hospital of Tamanrasset. One part of the blood taken from fingertip was used for blood smears and the second part was absorbed in filter paper for molecular diagnosis. Overall, the infection was detected by nested PCR in 63 samples versus 53 by direct examination. In addition, 11 mixed infections and 6 positive asymptomatic cases not detected by microscopy were diagnosed by PCR. Moreover, two negative samples in nested PCR were tested positive by direct examination. The molecular tool is more sensitive than the direct examination in detecting infra-microscopic parasitaemia and mixed infections...

Detection of Brucella spp. in milk from seronegative cows by real-time polymerase chain reaction in the region of Batna, Algeria

PubMed Central

Sabrina, Rabehi; Mossadak, Hamdi Taha; Bakir, Mamache; Asma, Meghezzi; Khaoula, Boushaba

2018-01-01

Aim: The aim of this study was to detect Brucella spp. DNA in milk samples collected from seronegative cows using the real-time polymerase chain reaction (PCR) assay for diagnosis of brucellosis in seronegative dairy cows to prevent transmission of disease to humans and to reduce economic losses in animal production. Materials and Methods: In this study, 65 milk samples were investigated for the detection of Brucella spp. The detection of the IS711 gene in all samples was done by real-time PCR assay by comparative cycle threshold method. Results: The results show that of the 65 DNA samples tested, 2 (3.08%) were positive for Brucella infection. The mean cyclic threshold values of IS711 real-time PCR test were 37.97 and 40.48, indicating a positive reaction. Conclusion: The results of the present study indicated that the real-time PCR appears to offer several advantages over serological tests. For this reason, the real-time PCR should be validated on representative numbers of Brucella-infected and free samples before being implemented in routine diagnosis in human and animal brucellosis for controlling this disease. PMID:29657430

Detecting the Presence of Nora Virus in "Drosophila" Utilizing Single Fly RT-PCR

ERIC Educational Resources Information Center

Munn, Bethany; Ericson, Brad; Carlson, Darby J.; Carlson, Kimberly A.

2015-01-01

A single fly RT-PCR protocol has recently been developed to detect the presence of the persistent, horizontally transmitted Nora virus in "Drosophila." Wild-caught flies from Ohio were tested for the presence of the virus, with nearly one-fifth testing positive. The investigation presented can serve as an ideal project for biology…

One-step cross-genogroup multiplex RT-qPCR with an internal control system for the detection of infectious pancreatic necrosis virus (IPNV).

PubMed

Hoferer, Marc; Braun, Anne; Skrypski, Julia; Bock, Sabine; Thalheim, Sabine; Sting, Reinhard

2017-09-01

Infectious pancreatic necrosis virus (IPNV) causes great losses in fish hatcheries world-wide. The detection of IPNV can be challenging in certain circumstances, particularly due to low viral load and the genetic variability of this RNA virus. For the first time, this project created a quantitative triplex real-time reverse transcription PCR (RT-qPCR), including an endogenous control system, for specific, sensitive and rapid detection of IPNV in routine diagnostics. Multiple sequence alignment of 46 nucleotide sequences of the segment A genome obtained from the NCBI database allowed the design of two RT-qPCR systems covering the IPNV genogroup 1 and genogroups 2-5, respectively. The completed triplex RT-qPCR including a salmonid-specific endogenous control showed high specificity and an analytical sensitivity of 20-40 oligonucleotide copies. Testing of dilution series of virus-loaded cell culture suspensions proved equality of the triplex RT-qPCR with virus detection in cell culture and a higher sensitivity than conventional RT-PCR in field samples. In comparative studies of a total of 77 field samples tested, 51 showed identical positive and 19 identical negative results in cell culture and the triplex RT-qPCR. However, seven other samples yielded positive results in the triplex RT-qPCR, but negative results in cell culture. Copyright © 2017 Elsevier B.V. All rights reserved.

Strip test for bedside detection of interleukin-6 in cervical secretions is predictive for impending preterm delivery.

PubMed

Trébéden, H; Goffinet, F; Kayem, G; Maillard, F; Lemoine, E; Cabrol, D; Weill, B; Batteux, F

2001-01-01

Inflammatory cytokines in amniotic fluid are markers of prematurity which could characterize preterm labour of infectious origin. To avoid amniocentesis, we analyzed IL-6, IL-8, IL-10, and IL-13 by RT-PCR in cervical secretions (CS) of 307 women with preterm labour. IL-6 was detected in 26.3% patients who delivered at less than 34 weeks (specificity: 95.8%). In addition, IL-6 was associated with delivery within 7 days (specificity: 91.6%). To render the detection more rapid and cheaper, a strip test was designed and evaluated comparatively with RT-PCR in 76 women. This bedside strip test was twice more sensitive than RT-PCR, with little decrease in specificity.

Detection limits of the strip test and PCR for genetically modified corn in Brazil.

PubMed

Nascimento, V E; Von Pinho, É V R; Von Pinho, R G; do Nascimento, A D

2012-08-16

Brazilian legislation establishes a labeling limit for products that contain more than 1% material from genetically modified organisms (GMOs). We assessed the sensitivity of the lateral flow strip test in detection of the GMO corn varieties Bt11 and MON810 and the specificity and sensitivity of PCR techniques for their detection. For the strip test, the GMO seeds were mixed with conventional seeds at levels of 0.2, 0.4 and 0.8% for Bt11, and 0.4, 0.8 and 1.6% for MON810. Three different methodologies were assessed and whole seeds, their endosperm and embryonic axis were used. For the PCR technique, the GMO seeds of each of the two varieties were mixed with conventional seeds at levels of 20, 10, 5, 2, 1, and 0.5%. The seeds were ground and the DNA extracted. For detection of the GMO material, specific primers were used for MON810 and Bt11 and maize zein as an endogenous control. The sensitivity of the strip test varied for both maize varieties and methodologies. The test was positive for Bt11 only at 0.8%, in contrast with the detection limit of 0.4% indicated by the manufacturer. In the multiplex PCR, the primers proved to be specific for the different varieties. These varieties were detected in samples with one GMO seed in 100. Thus, this technique proved to be efficient in detecting contaminations equal to or greater than 1%.

Comparison of the performance of three PCR assays for the detection and differentiation of Theileria orientalis genotypes.

PubMed

Perera, Piyumali K; Gasser, Robin B; Pulford, David J; Stevenson, Mark A; Firestone, Simon M; McFadden, Andrew M J; Jabbar, Abdul

2015-03-31

Oriental theileriosis is a tick-borne disease of bovines caused by the members of the Theileria orientalis complex. Recently, we developed a multiplexed tandem (MT) PCR to detect, differentiate and quantitate four genotypes (i.e., buffeli, chitose, ikeda and type 5) of T. orientalis. In this study, we used MT PCR to assess the prevalence and infection intensity of four T. orientalis genotypes in selected cattle herds that experienced oriental theileriosis outbreaks in New Zealand, and compared the sensitivities and specificities of MT PCR, PCR-high resolution melting (PCR-HRM) and a TaqMan qPCR. MT PCR, PCR-HRM analysis for T. orientalis and a TaqMan qPCR assay for ikeda genotype were employed to test 154 and 88 cattle blood samples from North (where oriental theileriosis outbreaks had occurred; designated as Group 1) and South (where no outbreaks had been reported; Group 2) Islands of New Zealand, respectively. Quantitative data from MT PCR assay were analyzed using generalized linear model and paired-sample t-test. The diagnostic specificity and sensitivity of the assays were estimated using a Bayesian latent class modeling approach. In Group 1, 99.4% (153/154) of cattle were test-positive for T. orientalis in both the MT PCR and PCR-HRM assays. The apparent prevalences of genotype ikeda in Group 1 were 87.6% (134/153) and 87.7% (135/154) using the MT PCR and Ikeda TaqMan qPCR assays, respectively. Using the MT PCR test, all four genotypes of T. orientalis were detected. The infection intensity estimated for genotype ikeda was significantly higher (P = 0.009) in severely anaemic cattle than in those without anaemia, and this intensity was significantly higher than that of buffeli (P < 0.001) in the former cattle. Bayesian latent class analysis showed that the diagnostic sensitivities (97.1-98.9%) and specificities (96.5-98.9%) of the three PCR assays were very comparable. The present findings show the advantages of using the MT PCR assay as a useful tool for in-depth epidemiological and transmission studies of T. orientalis worldwide.

Assessment of real-time PCR method for detection of EGFR mutation using both supernatant and cell pellet of malignant pleural effusion samples from non-small-cell lung cancer patients.

PubMed

Shin, Saeam; Kim, Juwon; Kim, Yoonjung; Cho, Sun-Mi; Lee, Kyung-A

2017-10-26

EGFR mutation is an emerging biomarker for treatment selection in non-small-cell lung cancer (NSCLC) patients. However, optimal mutation detection is hindered by complications associated with the biopsy procedure, tumor heterogeneity and limited sensitivity of test methodology. In this study, we evaluated the diagnostic utility of real-time PCR using malignant pleural effusion samples. A total of 77 pleural fluid samples from 77 NSCLC patients were tested using the cobas EGFR mutation test (Roche Molecular Systems). Pleural fluid was centrifuged, and separated cell pellets and supernatants were tested in parallel. Results were compared with Sanger sequencing and/or peptide nucleic acid (PNA)-mediated PCR clamping of matched tumor tissue or pleural fluid samples. All samples showed valid real-time PCR results in one or more DNA samples extracted from cell pellets and supernatants. Compared with other molecular methods, the sensitivity of real-time PCR method was 100%. Concordance rate of real-time PCR and Sanger sequencing plus PNA-mediated PCR clamping was 98.7%. We have confirmed that real-time PCR using pleural fluid had a high concordance rate compared to conventional methods, with no failed samples. Our data demonstrated that the parallel real-time PCR testing using supernatant and cell pellet could offer reliable and robust surrogate strategy when tissue is not available.

A novel and highly sensitive real-time nested RT-PCR assay in a single closed tube for detection of enterovirus.

PubMed

Shen, Xin-Xin; Qiu, Fang-Zhou; Zhao, Huai-Long; Yang, Meng-Jie; Hong, Liu; Xu, Song-Tao; Zhou, Shuai-Feng; Li, Gui-Xia; Feng, Zhi-Shan; Ma, Xue-Jun

2018-03-01

The sensitivity of qRT-PCR assay is not adequate for the detection of the samples with lower viral load, particularly in the cerebrospinal fluid (CSF) of patients. Here, we present the development of a highly sensitive real-time nested RT-PCR (RTN RT-PCR) assay in a single closed tube for detection of human enterovirus (HEV). The clinical performance of both RTN RT-PCR and qRT-PCR was also tested and compared using 140 CSF and fecal specimens. The sensitivities of RTN RT-PCR assay for EV71, Coxsackievirus A (CVA)16, CVA6 and CVA10 achieved 10 -8 dilution with a corresponding Ct value of 38.20, 36.45, 36.75, and 36.45, respectively, which is equal to traditional two-step nested RT-PCR assay and approximately 2-10-fold lower than that of qRT-PCR assay. The specificity of RTN RT-PCR assay was extensively analyzed insilico and subsequently verified using the reference isolates and clinical samples. Sixteen qRT-PCR-negative samples were detected by RTN RT-PCR and a variety of enterovirus serotypes was identified by sequencing of inner PCR products. We conclude RTN RT-PCR is more sensitive than qRT-PCR for the detection of HEV in clinical samples. Copyright © 2017 Elsevier Inc. All rights reserved.

Evaluation of detection methods for screening meat and poultry products for the presence of foodborne pathogens.

PubMed

Bohaychuk, Valerie M; Gensler, Gary E; King, Robin K; Wu, John T; McMullen, Lynn M

2005-12-01

Rapid and molecular technologies such as enzyme-linked immunosorbent assay (ELISA), PCR, and lateral flow immunoprecipitation can reduce the time and labor involved in screening food products for the presence of pathogens. These technologies were compared with conventional culture methodology for the detection of Salmonella, Campylobacter, Listeria, and Escherichia coli O157:H7 inoculated in raw and processed meat and poultry products. Recommended protocols were modified so that the same enrichment broths used in the culture methods were also used in the ELISA, PCR, and lateral flow immunoprecipitation assays. The percent agreement between the rapid technologies and culture methods ranged from 80 to 100% depending on the pathogen detected and the method used. ELISA, PCR, and lateral flow immunoprecipitation all performed well, with no statistical difference, compared with the culture method for the detection of E. coli O157:H7. ELISA performed better for the detection of Salmonella, with sensitivity and specificity rates of 100%. PCR performed better for the detection of Campylobacter jejuni, with 100% agreement to the culture method. PCR was highly sensitive for the detection of all the foodborne pathogens tested except Listeria monocytogenes. Although the lateral flow immunoprecipitation tests were statistically different from the culture methods for Salmonella and Listeria because of false-positive results, the tests did not produce any false negatives, indicating that this method would be suitable for screening meat and poultry products for these pathogens.

Development and Validation of a P-35S, T-nos, T-35S and P-FMV Tetraplex Real-time PCR Screening Method to Detect Regulatory Genes of Genetically Modified Organisms in Food.

PubMed

Eugster, Albert; Murmann, Petra; Kaenzig, Andre; Breitenmoser, Alda

2014-10-01

In routine analysis screening methods based on real-time PCR (polymerase chain reaction) are most commonly used for the detection of genetically modified (GM) plant material in food and feed. Screening tests are based on sequences frequently used for GM development, allowing the detection of a large number of GMOs (genetically modified organisms). Here, we describe the development and validation of a tetraplex real-time PCR screening assay comprising detection systems for the regulatory genes Cauliflower Mosaic Virus 35S promoter, Agrobacterium tumefaciens nos terminator, Cauliflower Mosaic Virus 35S terminator and Figwort Mosaic Virus 34S promoter. Three of the four primer and probe combinations have already been published elsewhere, whereas primers and probe for the 35S terminator have been developed in-house. Adjustment of primer and probe concentrations revealed a high PCR sensitivity with insignificant physical cross-talk between the four detection channels. The sensitivity of each PCR-system is sufficient to detect a GMO concentration as low as 0.05% of the containing respective element. The specificity of the described tetraplex is high when tested on DNA from GM maize, soy, rapeseed and tomato. We also demonstrate the robustness of the system by inter-laboratory tests. In conclusion, this method provides a sensitive and reliable screening procedure for the detection of the most frequently used regulatory elements present in GM crops either authorised or unauthorised for food.

Diagnostic performance of influenza viruses and RSV rapid antigen detection tests in children in tertiary care.

PubMed

Moesker, F M; van Kampen, J J A; Aron, G; Schutten, M; van de Vijver, D A M C; Koopmans, M P G; Osterhaus, A D M E; Fraaij, P L A

2016-06-01

Rapid antigen detection tests (RADTs) are increasingly used to detect influenza viruses and respiratory syncytial virus (RSV). However, their sensitivity and specificity are a matter of debate, challenging their clinical usefulness. Comparing diagnostic performances of BinaxNow Influenza AB(®) (BNI) and BinaxNow RSV(®) (BNR), to those of real-time reverse transcriptase PCR (RT-PCR), virus isolation and direct immunofluorescence (D-IF) in paediatric patients. Between November 2005 and September 2013, 521 nasal washings from symptomatic children (age <5 years) attending our tertiary care centre were tested, with a combination of the respective assays using RT-PCR as gold standard. Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of BNI were 69% (confidence interval [CI] [51-83]), 96% [94-97], 55% [39-70] and 98% [96-99] respectively. Of eleven false-negative samples, RT-PCR Ct-values were higher than all RT-PCR positive test results (27 vs 22, p=0.012). Of twenty false-positive samples, none were culture positive and two tested positive in D-IF. Sensitivity, specificity, PPV and NPV for BNR were 79% [73-85], 98% [96-99], 97% [93-99] and 88% [84-91]. Of the 42 false-negative samples the median Ct-value was higher than that of all RT-PCR positive samples (31 vs 23, p<0.0001). Five false-positive samples were detected. Three of these tested positive for RSV in virus isolation and D-IF. RADTs have a high specificity with BNR being superior to BNI. However, their relative low sensitivity limits their usefulness for clinical decision making in a tertiary care paediatric hospital. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

European Bat Lyssavirus in Scottish Bats

PubMed Central

Brookes, Sharon M.; Aegerter, James N.; Smith, Graham C.; Healy, Derek M.; Jolliffe, Tracey A.; Swift, Susan M.; Mackie, Iain J.; Pritchard, J. Stewart; Racey, Paul A.; Moore, Niall P.

2005-01-01

We report the first seroprevalence study of the occurrence of specific antibodies to European bat lyssavirus type 2 (EBLV-2) in Daubenton's bats. Bats were captured from 19 sites across eastern and southern Scotland. Samples from 198 Daubenton's bats, 20 Natterer's bats, and 6 Pipistrelle's bats were tested for EBLV-2. Blood samples (N = 94) were subjected to a modified fluorescent antibody virus neutralization test to determine antibody titer. From 0.05% to 3.8% (95% confidence interval) of Daubenton's bats were seropositive. Antibodies to EBLV-2 were not detected in the 2 other species tested. Mouth swabs (N = 218) were obtained, and RNA was extracted for a reverse transcription–polymerase chain reaction (RT-PCR). The RT-PCR included pan lyssavirus-primers (N gene) and internal PCR control primers for ribosomal RNA. EBLV-2 RNA was not detected in any of the saliva samples tested, and live virus was not detected in virus isolation tests. PMID:15829196

Development of a PCR Assay for the Detection of Spironucleus muris

PubMed Central

Jackson, Glenn A; Livingston, Robert S; Riley, Lela K; Livingston, Beth A; Franklin, Craig L

2013-01-01

Spironucleus muris is a protozoan that can colonize the intestinal tract of many rodent species. Although its effects on animal health and research are debated, S. muris is often included on exclusion lists for rodent facilities. Common diagnostic tests for S. muris are insensitive and typically are performed at postmortem examination. We sought to develop a PCR-based diagnostic test with sufficient sensitivity and specificity for use on fecal samples from live rodents. We designed and optimized a PCR assay that targeted the 16S-like rRNA gene of S. muris. The assay was highly specific, given that samples from mice contaminated with S. muris were PCR positive, whereas samples from mice contaminated with other protozoa were negative. The assay also was highly sensitive, detecting as few as 5 template copies per microliter diluent. All mice positive for S. muris on postmortem exams also were positive by fecal PCR. Moreover, S. muris was detected by PCR in mice negative by postmortem examination but from colonies known to be contaminated as well as in rats and hamsters. To assess protozoal loads in mice of differing ages, the PCR assay was adapted to a quantitative format. Fecal loads of S. muris were highest in 4-wk-old mice and declined with age. The PCR assay developed promises to be a highly specific antemortem diagnostic assay with higher sensitivity than that of existing postmortem tests. PMID:23562099

Rapid identification of HPV 16 and 18 by multiplex nested PCR-immunochromatographic test.

PubMed

Kuo, Yung-Bin; Li, Yi-Shuan; Chan, Err-Cheng

2015-02-01

Human papillomavirus (HPV) types 16 and 18 are known to be high-risk viruses that cause cervical cancer. An HPV rapid testing kit that could help physicians to make early and more informed decisions regarding patient care is needed urgently but not yet available. This study aimed to develop a multiplex nested polymerase chain reaction-immunochromatographic test (PCR-ICT) for the rapid identification of HPV 16 and 18. A multiplex nested PCR was constructed to amplify the HPV 16 and 18 genotype-specific L1 gene fragments and followed by ICT which coated with antibodies to identify rapidly the different PCR products. The type-specific gene regions of high-risk HPV 16 and 18 could be amplified successfully by multiplex nested PCR at molecular sizes of approximately 99 and 101bp, respectively. The capture antibodies raised specifically against the moleculars labeled on the PCR products could be detected simultaneously both HPV 16 and 18 in one strip. Under optimal conditions, this PCR-ICT assay had the capability to detect HPV in a sample with as low as 100 copies of HPV viral DNA. The PCR-ICT system has the advantage of direct and simultaneous detection of two high-risk HPV 16 and 18 DNA targets in one sample, which suggested a significant potential of this assay for clinical application. Copyright © 2014. Published by Elsevier B.V.

Development, application, and results of routine monitoring of Marek's disease virus in broiler house dust using real-time quantitative PCR.

PubMed

Walkden-Brown, Stephen W; Islam, A F Aminul; Groves, Peter J; Rubite, Ambrosio; Sharpe, Sue M; Burgess, Susan K

2013-06-01

Results are presented from four studies between 2002 and 2011 into the feasibility of routinely monitoring Marek's disease virus serotype 1 (MDV-1) in broiler house dust using real-time quantitative PCR (qPCR) measurement. Study 1 on two farms showed that detection of MDV-1 occurred earlier on average in dust samples tested using qPCR than standard PCR and in spleen samples from five birds per shed assayed for MDV-1 by qPCR or standard PCR. DNA quality following extraction from dust had no effect on detection of MDV-1. Study 2 demonstrated that herpesvirus of turkeys (HVT) and MDV serotype 2 (MDV-2) in addition to MDV-1 could be readily amplified from commercial farm dust samples, often in mixtures. MDV-2 was detected in 11 of 20 samples despite the absence of vaccination with this serotype. Study 3 investigated the reproducibility and sensitivity of the qPCR test and the presence of inhibitors in the samples. Samples extracted and amplified in triplicate showed a high level of reproducibility except at very low levels of virus near the limit of detection. Mixing of samples prior to extraction provided results consistent with the proportions in the mixture. Tests for inhibition showed that if the template contained DNA in the range 0.5-20 ng/microl no inhibition of the reaction was detectable. The sensitivity of the tests in terms of viral copy number (VCN) per milligram of dust was calculated to be in the range 24-600 VCN/mg for MDV-1, 48-1200 VCN/mg for MDV-2, and 182-4560 VCN/mg for HVT. In study 4 the results of 1976 commercial tests carried out for one company were analyzed. Overall 23.1% of samples were positive for MDV-1, 26.1% in unvaccinated and 16.4% in vaccinated chickens. There was marked regional and temporal variation in the proportion of positive samples and the MDV-1 load. The tests were useful in formulating Marek's disease vaccination strategies. The number of samples submitted has increased recently, as has the incidence of positive samples. These studies provide strong evidence that detection and quantitation of MDV-1, HVT, and MDV-2 in poultry house dust using qPCR is robust, sensitive, reproducible, and meaningful, both biologically and commercially. Tactical vaccination based on monitoring of MDV-1 rather than routine vaccination may reduce selection pressure for increased virulence in MDV-1.

Evaluation of a Turbidimetric β-d-Glucan Test for Detection of Pneumocystis jirovecii Pneumonia.

PubMed

Dichtl, Karl; Seybold, Ulrich; Wagener, Johannes

2018-07-01

Currently, diagnosis of Pneumocystis jirovecii pneumonia (PJP) relies on analysis of lower respiratory specimens, either by microscopy or quantitative real-time PCR (qPCR). Thus, bronchoscopy is required, which is associated with increased risk of respiratory failure. We assessed the value of noninvasive serologic β-d-glucan (BDG) testing for laboratory diagnosis of PJP using a newly available turbidimetric assay. We identified 73 cases of PJP with positive qPCR results from lower respiratory specimens for Pneumocystis and serology samples dating from 1 week before to 4 weeks after qPCR. In addition, 25 sera from controls with suspected PJP but specimens negative for Pneumocystis by qPCR were identified. Sera were tested with a turbidimetric BDG assay (Fujifilm Wako Chemicals Europe GmbH, Neuss, Germany), using an 11-pg/ml cutoff. Sensitivity and specificity were calculated based on qPCR test results as a reference. The turbidimetric BDG assay identified 63/73 patients with positive or slightly positive qPCR tests for an overall sensitivity of 86%; after exclusion of cases with only slightly positive qPCR results, sensitivity was 91%. No correlation between serum BDG levels and respiratory specimen DNA levels was found. Serologic BDG testing was negative in 25/25 controls with negative qPCR for a specificity of 100% using the predefined cutoff. In 22/25 samples (88%), no BDG was detected. Serologic BDG testing using the turbidimetric assay showed high sensitivity and specificity compared to qPCR of lower respiratory specimens for the diagnosis of PJP. Both turnover time and test performance will allow clinicians to delay or in some cases forego bronchoscopy. Copyright © 2018 American Society for Microbiology.

Novel approach based on one-tube nested PCR and a lateral flow strip for highly sensitive diagnosis of tuberculous meningitis

PubMed Central

Sun, Yajuan; Chen, Jiajun; Li, Jia; Xu, Yawei; Jin, Hui; Xu, Na; Yin, Rui

2017-01-01

Rapid and sensitive detection of Mycobacterium tuberculosis (M. Tb) in cerebrospinal fluid is crucial in the diagnosis of tuberculous meningitis (TBM), but conventional diagnostic technologies have limited sensitivity and specificity or are time-consuming. In this work, a novel, highly sensitive molecular diagnostic method, one-tube nested PCR-lateral flow strip test (OTNPCR-LFST), was developed for detecting M. tuberculosis. This one-tube nested PCR maintains the sensitivity of conventional two-step nested PCR and reduces both the chance of cross-contamination and the time required for analysis. The PCR product was detected by a lateral flow strip assay, which provided a basis for migration of the test to a point-of-care (POC) microfluidic format. The developed assay had an improved sensitivity compared with traditional PCR, and the limit of detection was up to 1 fg DNA isolated from M. tuberculosis. The assay was also specific for M. tuberculosis, and no cross-reactions were found in other non-target bacteria. The application of this technique to clinical samples was successfully evaluated, and OTNPCR-LFST showed 89% overall sensitivity and 100% specificity for TBM patients. This one-tube nested PCR-lateral flow strip assay is useful for detecting M. tuberculosis in TBM due to its rapidity, high sensitivity and simple manipulation. PMID:29084241

Improved detection of Burkholderia pseudomallei from non-blood clinical specimens using enrichment culture and PCR: narrowing diagnostic gap in resource-constrained settings.

PubMed

Tellapragada, Chaitanya; Shaw, Tushar; D'Souza, Annet; Eshwara, Vandana Kalwaje; Mukhopadhyay, Chiranjay

2017-07-01

To evaluate the diagnostic utility of enrichment culture and PCR for improved case detection rates of non-bacteraemic form of melioidosis in limited resource settings. Clinical specimens (n = 525) obtained from patients presenting at a tertiary care hospital of South India with clinical symptoms suggestive of community-acquired pneumonia, lower respiratory tract infections, superficial or internal abscesses, chronic skin ulcers and bone or joint infections were tested for the presence of Burkholderia pseudomallei using conventional culture (CC), enrichment culture (EC) and PCR. Sensitivity, specificity, positive and negative predictive values of CC and PCR were initially deduced using EC as the gold standard method. Further, diagnostic accuracies of all the three methods were analysed using Bayesian latent class modelling (BLCM). Detection rates of B. pseudomallei using CC, EC and PCR were 3.8%, 5.3% and 6%, respectively. Diagnostic sensitivities and specificities of CC and PCR were 71.4, 98.4% and 100 and 99.4%, respectively in comparison with EC as the gold standard test. With Bayesian latent class modelling, EC and PCR demonstrated sensitivities of 98.7 and 99.3%, respectively, while CC showed a sensitivity of 70.3% for detection of B. pseudomallei. An increase of 1.6% (95% CI: 1.08-4.32%) in the case detection rate of melioidosis was observed in the study population when EC and/or PCR were used in adjunct to the conventional culture technique. Our study findings underscore the diagnostic superiority of enrichment culture and/or PCR over conventional microbiological culture for improved case detection of melioidosis from non-blood clinical specimens. © 2017 John Wiley & Sons Ltd.

Design and verification of a highly reliable Linear-After-The-Exponential PCR (LATE-PCR) assay for the detection of African swine fever virus.

PubMed

Ronish, B; Hakhverdyan, M; Ståhl, K; Gallardo, C; Fernandez-Pinero, J; Belák, S; Leblanc, N; Wangh, L

2011-03-01

African swine fever virus (ASFV) is a highly pathogenic DNA virus that is the causative agent of African swine fever (ASF), an infectious disease of domestic and wild pigs of all breeds and ages, causing a range of syndromes. Acute disease is characterized by high fever, haemorrhages in the reticuloendothelial system, and a high mortality rate. A powerful novel diagnostic assay based on the Linear-After-The-Exponential-PCR (LATE-PCR) principle was developed to detect ASFV. LATE-PCR is an advanced form of asymmetric PCR which results in direct amplification of large amount of single-stranded DNA. Fluorescent readings are acquired using endpoint analysis after PCR amplification. Amplification of the correct product is verified by melting curve analysis. The assay was designed to amplify the VP72 gene of ASFV genome. Nineteen ASFV DNA cell culture virus strains and three tissue samples (spleen, tonsil, and liver) from infected experimental pigs were tested. Virus was detected in all of the cell culture and tissue samples. None of five ASFV-related viruses tested produced a positive signal, demonstrating the high specificity of the assay. The sensitivity of the LATE-PCR assay was determined in two separate real-time monoplex reactions using samples of synthetic ASFV and synthetic control-DNA targets that were diluted serially from 10⁹ to 1 initial copies per reaction. The detection limit was 1 and 10 copies/reaction, respectively. The sensitivity of the assay was also tested in a duplex end-point reactions comprised of a constant level of 150 copies of synthetic control-DNA and a clinical sample of spleen tissue diluted serially from 10⁻¹ to 10⁻⁵. The detection limit was 10⁻⁵ dilution which corresponds to approximately 1 copy/reaction. Since the assay is designed to be used in either laboratory settings or in a portable PCR machine (Bio-Seeq Portable Veterinary Diagnostics Laboratory; Smiths Detection, Watford UK), the LATE-PCR provides a robust and novel tool for the diagnosis of ASF both in the laboratory and in the field. Copyright © 2010 Elsevier B.V. All rights reserved.

A quantitative TaqMan PCR assay for the detection of Ureaplasma diversum.

PubMed

Marques, Lucas M; Amorim, Aline T; Martins, Hellen Braga; Rezende, Izadora Souza; Barbosa, Maysa Santos; Lobão, Tassia Neves; Campos, Guilherme B; Timenetsky, Jorge

2013-12-27

Ureaplasma diversum in veterinary studies is an undesirable microbe, which may cause infection in bulls and may result in seminal vesiculitis, balanopostitis, and alterations in spermatozoids, whereas in cows, it may cause placentitis, fetal alveolitis, abortion, and birth of weak calves. U. diversum is released through organic secretions, especially semen, preputial and vaginal mucus, conjunctival secretion, and milk. The aim of the present study was to develop a TaqMan probe, highly sensitive and specific quantitative PCR (qPCR) assay for the detection and quantification of U. diversum from genital swabs of bovines. Primers and probes specific to U. diversum 16S rRNA gene were designed. The specificity, detection limit, intra- and inter-assay variability of qPCR to detect this ureaplasma was compared with the results of the conventional PCR assay (cPCR). Swabs of vaginal mucus from 169 cows were tested. The qPCR assay detected as few as 10 copies of U. diversum and was 100-fold more sensitive than the cPCR. No cross-reactivity with other Mollicutes or eubacteria was observed. U. diversum was detected in 79 swabs (46.42%) by qPCR, while using cPCR it was detected in 42 (25%) samples. The difference in cPCR and qPCR ureaplasma detection between healthy and sick animals was not statistically significant. But the U. diversum load in samples from animals with genital disorders was higher than in healthy animals. The qPCR assay developed herein is highly sensitive and specific for the detection and quantification of U. diversum in vaginal bovine samples. Copyright © 2013. Published by Elsevier B.V.

Application of PCR to a clinical and environmental investigation of a case of equine botulism.

PubMed

Szabo, E A; Pemberton, J M; Gibson, A M; Thomas, R J; Pascoe, R R; Desmarchelier, P M

1994-08-01

PCR for the detection of botulinum neurotoxin gene types A to E was used in the investigation of a case of equine botulism. Samples from a foal diagnosed with toxicoinfectious botulism in 1985 were reanalyzed by PCR and the mouse bioassay in conjunction with an environmental survey. Neurotoxin B was detected by mouse bioassay in culture enrichments of serum, spleen, feces, and intestinal contents. PCR results compared well with mouse bioassay results, detecting type B neurotoxin genes in these samples and also in a liver sample. Other neurotoxin types were not detected by either test. Clostridium botulinum type B was shown to be prevalent in soils collected from the area in which the foal was raised. Four methods were used to test for the presence of botulinum neurotoxin-producing organisms in 66 soil samples taken within a 5-km radius: PCR and agarose gel electrophoresis (types A to E), PCR and an enzyme-linked assay (type B), hybridization of crude alkaline cell lysates with a type B-specific probe, and the mouse bioassay (all types). Fewer soil samples were positive for C. botulinum type B by the mouse bioassay (15%) than by any of the DNA-based detection systems. Hybridization of a type B-specific probe to DNA dot blots (26% of the samples were positive) and PCR-enzyme-linked assay (77% of the samples were positive) were used for the rapid analysis of large numbers of samples, with sensitivity limits of 3 x 10(6) and 3,000 cells, respectively. Conventional detection of PCR products by gel electrophoresis was the most sensitive method (300-cell limit), and in the present environmental survey, neurotoxin B genes only were detected in 94% of the samples.

Application of PCR to a clinical and environmental investigation of a case of equine botulism.

PubMed Central

Szabo, E A; Pemberton, J M; Gibson, A M; Thomas, R J; Pascoe, R R; Desmarchelier, P M

1994-01-01

PCR for the detection of botulinum neurotoxin gene types A to E was used in the investigation of a case of equine botulism. Samples from a foal diagnosed with toxicoinfectious botulism in 1985 were reanalyzed by PCR and the mouse bioassay in conjunction with an environmental survey. Neurotoxin B was detected by mouse bioassay in culture enrichments of serum, spleen, feces, and intestinal contents. PCR results compared well with mouse bioassay results, detecting type B neurotoxin genes in these samples and also in a liver sample. Other neurotoxin types were not detected by either test. Clostridium botulinum type B was shown to be prevalent in soils collected from the area in which the foal was raised. Four methods were used to test for the presence of botulinum neurotoxin-producing organisms in 66 soil samples taken within a 5-km radius: PCR and agarose gel electrophoresis (types A to E), PCR and an enzyme-linked assay (type B), hybridization of crude alkaline cell lysates with a type B-specific probe, and the mouse bioassay (all types). Fewer soil samples were positive for C. botulinum type B by the mouse bioassay (15%) than by any of the DNA-based detection systems. Hybridization of a type B-specific probe to DNA dot blots (26% of the samples were positive) and PCR-enzyme-linked assay (77% of the samples were positive) were used for the rapid analysis of large numbers of samples, with sensitivity limits of 3 x 10(6) and 3,000 cells, respectively. Conventional detection of PCR products by gel electrophoresis was the most sensitive method (300-cell limit), and in the present environmental survey, neurotoxin B genes only were detected in 94% of the samples. Images PMID:7989554

Rapid detection of pathological mutations and deletions of the haemoglobin beta gene (HBB) by High Resolution Melting (HRM) analysis and Gene Ratio Analysis Copy Enumeration PCR (GRACE-PCR).

PubMed

Turner, Andrew; Sasse, Jurgen; Varadi, Aniko

2016-10-19

Inherited disorders of haemoglobin are the world's most common genetic diseases, resulting in significant morbidity and mortality. The large number of mutations associated with the haemoglobin beta gene (HBB) makes gene scanning by High Resolution Melting (HRM) PCR an attractive diagnostic approach. However, existing HRM-PCR assays are not able to detect all common point mutations and have only a very limited ability to detect larger gene rearrangements. The aim of the current study was to develop a HBB assay, which can be used as a screening test in highly heterogeneous populations, for detection of both point mutations and larger gene rearrangements. The assay is based on a combination of conventional HRM-PCR and a novel Gene Ratio Analysis Copy Enumeration (GRACE) PCR method. HRM-PCR was extensively optimised, which included the use of an unlabelled probe and incorporation of universal bases into primers to prevent interference from common non-pathological polymorphisms. GRACE-PCR was employed to determine HBB gene copy numbers relative to a reference gene using melt curve analysis to detect rearrangements in the HBB gene. The performance of the assay was evaluated by analysing 410 samples. A total of 44 distinct pathological genotypes were detected. In comparison with reference methods, the assay has a sensitivity of 100 % and a specificity of 98 %. We have developed an assay that detects both point mutations and larger rearrangements of the HBB gene. This assay is quick, sensitive, specific and cost effective making it suitable as an initial screening test that can be used for highly heterogeneous cohorts.

Real-time PCR assays for detection of Brucella spp. and the identification of genotype ST27 in bottlenose dolphins (Tursiops truncatus).

PubMed

Wu, Qingzhong; McFee, Wayne E; Goldstein, Tracey; Tiller, Rebekah V; Schwacke, Lori

2014-05-01

Rapid detection of Brucella spp. in marine mammals is challenging. Microbiologic culture is used for definitive diagnosis of brucellosis, but is time consuming, has low sensitivity and can be hazardous to laboratory personnel. Serological methods can aid in diagnosis, but may not differentiate prior exposure versus current active infection and may cross-react with unrelated Gram-negative bacteria. This study reports a real-time PCR assay for the detection of Brucella spp. and application to screen clinical samples from bottlenose dolphins stranded along the coast of South Carolina, USA. The assay was found to be 100% sensitive for the Brucella strains tested, and the limit of detection was 0.27fg of genomic DNA from Brucella ceti B1/94 per PCR volume. No amplification was detected for the non-Brucella pathogens tested. Brucella DNA was detected in 31% (55/178) of clinical samples tested. These studies indicate that the real-time PCR assay is highly sensitive and specific for the detection of Brucella spp. in bottlenose dolphins. We also developed a second real-time PCR assay for rapid identification of Brucella ST27, a genotype that is associated with human zoonotic infection. Positive results were obtained for Brucella strains which had been identified as ST27 by multilocus sequence typing. No amplification was found for other Brucella strains included in this study. ST27 was identified in 33% (18/54) of Brucella spp. DNA-positive clinical samples. To our knowledge, this is the first report on the use of a real-time PCR assay for identification of Brucella genotype ST27 in marine mammals. Copyright © 2014 Elsevier B.V. All rights reserved.

Development of molecular tests for the detection of ILAR and latent viruses in fruit trees.

PubMed

Roussel, S; Kummert, J; Dutrecq, O; Lepoivre, P; Jijakli, M H

2004-01-01

The detection throughout the year of latent and ILAR viruses in fruit tress by classical serological tests appear to be unreliable. We have developed RT-PCR tests for a reliable detection of latent and ILAR viruses in fruit trees. These assays were then simplified to allow the direct use of crude plant extracts instead of total RNA preparations, and the analyses of pooled samples. In this way, such RT-PCR protocols are suitable for a routine diagnosis of latent and ILAR viruses in fruit tree certification.

Evaluation of the hyplex® TBC PCR test for detection of Mycobacterium tuberculosis complex in clinical samples

PubMed Central

2010-01-01

Background Tuberculosis (TB) is one of the major public health concerns worldwide. The detection of the pathogen Mycobacterium tuberculosis complex (MTBC) as early as possible has a great impact on the effective control of the spread of the disease. In our study, we evaluated the hyplex® TBC PCR test (BAG Health Care GmbH), a novel assay using a nucleic acid amplification technique (NAAT) with reverse hybridisation and ELISA read out for the rapid detection of M. tuberculosis directly in clinical samples. Results A total of 581 respiratory and non-respiratory specimens from our pneumological hospital and the National TB Institute of Uzbekistan were used for the evaluation of the PCR assay. Of these, 292 were classified as TB samples and 289 as non-TB samples based on the results of the TB cultures as reference method. The PCR results were initially used to optimise the cut-off value of the hyplex® TBC test system by means of a ROC analysis. The overall sensitivity of the assay was determined to be 83.1%. In smear-positive TB samples, the sensitivity of the hyplex® TBC PCR test was estimated to 93.4% versus 45.1% in smear-negative samples. The specificity of the test was 99.25%. Of the two specimens (0.75%) with false-positive PCR results, one yielded a culture positive for non-tuberculous mycobacteria. Based on the assumption of a prevalence of 8% TB positives among the samples in our diagnostic TB laboratory, the positive and negative predictive values were estimated to 90.4% and 98.5%, respectively. Conclusions The hyplex® TBC PCR test is an accurate NAAT assay for a rapid and reliable detection of M. tuberculosis in various respiratory and non-respiratory specimens. Compared to many other conventional NAAT assays, the hyplex® TBC PCR test is in a low price segment which makes it an attractive option for developing and emerging countries with high TB burdens. PMID:20356361

Salmonella detection in poultry samples. Comparison of two commercial real-time PCR systems with culture methods for the detection of Salmonella spp. in environmental and fecal samples of poultry.

PubMed

Sommer, D; Enderlein, D; Antakli, A; Schönenbrücher, H; Slaghuis, J; Redmann, T; Lierz, M

2012-01-01

The efficiency of two commercial PCR methods based on real-time technology, the foodproof® Salmonella detection system and the BAX® PCR Assay Salmonella system was compared to standardized culture methods (EN ISO 6579:2002 - Annex D) for the detection of Salmonella spp. in poultry samples. Four sample matrices (feed, dust, boot swabs, feces) obtained directly from poultry flocks, as well as artificially spiked samples of the same matrices, were used. All samples were tested for Salmonella spp. using culture methods first as the gold standard. In addition samples spiked with Salmonella Enteridis were tested to evaluate the sensitivity of both PCR methods. Furthermore all methods were evaluated in an annual ring-trial of the National Salmonella Reference Laboratory of Germany. Salmonella detection in the matrices feed, dust and boot swabs were comparable in both PCR systems whereas the results from feces differed markedly. The quality, especially the freshness, of the fecal samples had an influence on the sensitivity of the real-time PCR and the results of the culture methods. In fresh fecal samples an initial spiking level of 100cfu/25g Salmonella Enteritidis was detected. Two-days-dried fecal samples allowed the detection of 14cfu/25g. Both real- time PCR protocols appear to be suitable for the detection of Salmonella spp. in all four matrices. The foodproof® system detected eight samples more to be positive compared to the BAX® system, but had a potential false positive result in one case. In 7-days-dried samples none of the methods was able to detect Salmonella likely through letal cell damage. In general the advantage of PCR analyses over the culture method is the reduction of working time from 4-5 days to only 2 days. However, especially for the analysis of fecal samples official validation should be conducted according to the requirement of EN ISO6579:2002 - Annex D.

Recombinase Polymerase Amplification Compared to Real-Time Polymerase Chain Reaction Test for the Detection of Fasciola hepatica in Human Stool

PubMed Central

Cabada, Miguel M.; Malaga, Jose L.; Castellanos-Gonzalez, Alejandro; Bagwell, Kelli A.; Naeger, Patrick A.; Rogers, Hayley K.; Maharsi, Safa; Mbaka, Maryann; White, A. Clinton

2017-01-01

Fasciola hepatica is the most widely distributed trematode infection in the world. Control efforts may be hindered by the lack of diagnostic capacity especially in remote endemic areas. Polymerase chain reaction (PCR)–based methods offer high sensitivity and specificity but require expensive technology. However, the recombinase polymerase amplification (RPA) is an efficient isothermal method that eliminates the need for a thermal cycler and has a high deployment potential to resource-limited settings. We report on the characterization of RPA and PCR tests to detect Fasciola infection in clinical stool samples with low egg burdens. The sensitivity of the RPA and PCR were 87% and 66%, respectively. Both tests were 100% specific showing no cross-reactivity with trematode, cestode, or nematode parasites. In addition, RPA and PCR were able to detect 47% and 26% of infections not detected by microscopy, respectively. The RPA adapted to a lateral flow platform was more sensitive than gel-based detection of the reaction products. In conclusion, the Fasciola RPA is a highly sensitive and specific test to diagnose chronic infection using stool samples. The Fasciola RPA lateral flow has the potential for deployment to endemic areas after further characterization. PMID:27821691

Ureaplasma parvum prosthetic joint infection detected by PCR.

PubMed

Farrell, John J; Larson, Joshua A; Akeson, Jeffrey W; Lowery, Kristin S; Rounds, Megan A; Sampath, Rangarajan; Bonomo, Robert A; Patel, Robin

2014-06-01

We describe the first reported case of Ureaplasma parvum prosthetic joint infection (PJI) detected by PCR. Ureaplasma species do not possess a cell wall and are usually associated with colonization and infection of mucosal surfaces (not prosthetic material). U. parvum is a relatively new species name for certain serovars of Ureaplasma urealyticum, and PCR is useful for species determination. Our patient presented with late infection of his right total knee arthroplasty. Intraoperative fluid and tissue cultures and pre- and postoperative synovial fluid cultures were all negative. To discern the pathogen, we employed PCR coupled with electrospray ionization mass spectrometry (PCR/ESI-MS). Our patient's failure to respond to empirical antimicrobial treatment and our previous experience with PCR/ESI-MS in culture-negative cases of infection prompted us to use this approach over other diagnostic modalities. PCR/ESI-MS detected U. parvum in all samples. U. parvum-specific PCR testing was performed on all synovial fluid samples to confirm the U. parvum detection. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Detection of the enzymatically-active polyhydroxyalkanoate synthase subunit gene, phaC, in cyanobacteria via colony PCR.

PubMed

Lane, Courtney E; Benton, Michael G

2015-12-01

A colony PCR-based assay was developed to rapidly determine if a cyanobacterium of interest contains the requisite genetic material, the PHA synthase PhaC subunit, to produce polyhydroxyalkanoates (PHAs). The test is both high throughput and robust, owing to an extensive sequence analysis of cyanobacteria PHA synthases. The assay uses a single detection primer set and a single reaction condition across multiple cyanobacteria strains to produce an easily detectable positive result - amplification via PCR as evidenced by a band in electrophoresis. In order to demonstrate the potential of the presence of phaC as an indicator of a cyanobacteria's PHA accumulation capabilities, the ability to produce PHA was assessed for five cyanobacteria with a traditional in vivo PHA granule staining using an oxazine dye. The confirmed in vivo staining results were then compared to the PCR-based assay results and found to be in agreement. The colony PCR assay was capable of successfully detecting the phaC gene in all six of the diverse cyanobacteria tested which possessed the gene, while exhibiting no undesired product formation across the nine total cyanobacteria strains tested. The colony PCR quick prep provides sufficient usable DNA template such that this assay could be readily expanded to assess multiple genes of interest simultaneously. Copyright © 2015 Elsevier Ltd. All rights reserved.

Detection of the aerolysin gene in Aeromonas hydrophila by the polymerase chain reaction.

PubMed Central

Pollard, D R; Johnson, W M; Lior, H; Tyler, S D; Rozee, K R

1990-01-01

Synthetic oligonucleotide primers were used in a polymerase chain reaction (PCR) technique to detect the gene for aerolysin in strains of Aeromonas hydrophila and to screen for identical genes in A. caviae, A. sobria, and A. veronii isolated from patients with diarrheal disease. Primers targeted a 209-bp fragment of the aer gene coding for the beta-hemolysin and detected template DNA only in the PCR using nucleic acid (NA) from hemolytic strains of A. hydrophila which were also cytotoxic to Vero and CHO cells and enterotoxic in suckling-mouse assays. PCR amplification of NA from hemolytic A. sobria or nonhemolytic A. hydrophila and A. caviae strains was consistently negative. Primer specificity was determined in the PCR by using NA extracted from 56 strains of bacteria, including hemolytic Escherichia coli and Listeria monocytogenes as well as several recognized enteric pathogens defined in terms of their toxigenicity. The detection limit for the aerolysin gene by PCR amplification was 1 ng of total NA. The PCR clearly identified aerolysin-producing strains of A. hydrophila and may have application as a species-specific virulence test because other hemolytic Aeromonas species tested were negative. Images PMID:2254423

The IRIDICA PCR/Electrospray Ionization-Mass Spectrometry Assay on Bronchoalveolar Lavage for Bacterial Etiology in Mechanically Ventilated Patients with Suspected Pneumonia.

PubMed

Strålin, Kristoffer; Ehn, Fredrik; Giske, Christian G; Ullberg, Måns; Hedlund, Jonas; Petersson, Johan; Spindler, Carl; Özenci, Volkan

2016-01-01

We studied the diagnostic performance of the IRIDICA PCR/electrospray ionization-mass spectrometry (PCR/ESI-MS) assay applied on bronchoalveolar lavage (BAL) samples, from 51 mechanically ventilated patients with suspected pneumonia, in a prospective study. In 32 patients with X-ray verified pneumonia, PCR/ESI-MS was positive in 66% and BAL culture was positive in 38% (p = 0.045), and either of the methods was positive in 69%. The following BAL result combinations were noted: PCR/ESI-MS+/culture+, 34%; PCR/ESI-MS+/culture-, 31%; PCR/ESI-MS-/culture+, 3.1%; PCR/ESI-MS-/culture-, 31%; kappa 0.36 (95% confidence interval (CI), 0.10-0.63). In pneumonia patients without prior antibiotic treatment, optimal agreement was noted with 88% PCR/ESI-MS+/culture+ and 12% PCR/ESI-MS-/culture- (kappa 1.0). However, in patients with prior antibiotic treatment, the test agreement was poor (kappa 0.16; 95% CI, -0.10-0.44), as 10 patients were PCR/ESI-MS+/culture-. In 8/10 patients the pathogens detected by PCR/ESI-MS could be detected by other conventional tests or PCR tests on BAL. Compared with BAL culture, PCR/ESI-MS showed specificities and negative predictive values of ≥87% for all individual pathogens, an overall sensitivity of 77% and positive predictive value (PPV) of 42%. When other conventional tests and PCR tests were added to the reference standard, the overall PPV increased to 87%. The PCR/ESI-MS semi-quantitative level tended to be higher for PCR/ESI-MS positive cases with pneumonia compared with cases without pneumonia (p = 0.074). In conclusion, PCR/ESI-MS applied on BAL showed a promising performance and has potential to be clinically useful in mechanically ventilated patients with suspected pneumonia. The usefulness of the method for establishment of pneumonia etiology and selection of antibiotic therapy should be further studied.

The IRIDICA PCR/Electrospray Ionization–Mass Spectrometry Assay on Bronchoalveolar Lavage for Bacterial Etiology in Mechanically Ventilated Patients with Suspected Pneumonia

PubMed Central

Ehn, Fredrik; Giske, Christian G.; Ullberg, Måns; Hedlund, Jonas; Petersson, Johan; Spindler, Carl; Özenci, Volkan

2016-01-01

We studied the diagnostic performance of the IRIDICA PCR/electrospray ionization–mass spectrometry (PCR/ESI-MS) assay applied on bronchoalveolar lavage (BAL) samples, from 51 mechanically ventilated patients with suspected pneumonia, in a prospective study. In 32 patients with X-ray verified pneumonia, PCR/ESI-MS was positive in 66% and BAL culture was positive in 38% (p = 0.045), and either of the methods was positive in 69%. The following BAL result combinations were noted: PCR/ESI-MS+/culture+, 34%; PCR/ESI-MS+/culture-, 31%; PCR/ESI-MS-/culture+, 3.1%; PCR/ESI-MS-/culture-, 31%; kappa 0.36 (95% confidence interval (CI), 0.10–0.63). In pneumonia patients without prior antibiotic treatment, optimal agreement was noted with 88% PCR/ESI-MS+/culture+ and 12% PCR/ESI-MS-/culture- (kappa 1.0). However, in patients with prior antibiotic treatment, the test agreement was poor (kappa 0.16; 95% CI, -0.10–0.44), as 10 patients were PCR/ESI-MS+/culture-. In 8/10 patients the pathogens detected by PCR/ESI-MS could be detected by other conventional tests or PCR tests on BAL. Compared with BAL culture, PCR/ESI-MS showed specificities and negative predictive values of ≥87% for all individual pathogens, an overall sensitivity of 77% and positive predictive value (PPV) of 42%. When other conventional tests and PCR tests were added to the reference standard, the overall PPV increased to 87%. The PCR/ESI-MS semi-quantitative level tended to be higher for PCR/ESI-MS positive cases with pneumonia compared with cases without pneumonia (p = 0.074). In conclusion, PCR/ESI-MS applied on BAL showed a promising performance and has potential to be clinically useful in mechanically ventilated patients with suspected pneumonia. The usefulness of the method for establishment of pneumonia etiology and selection of antibiotic therapy should be further studied. PMID:27463099

Automated Nucleic Acid Extraction Systems for Detecting Cytomegalovirus and Epstein-Barr Virus Using Real-Time PCR: A Comparison Study Between the QIAsymphony RGQ and QIAcube Systems.

PubMed

Kim, Hanah; Hur, Mina; Kim, Ji Young; Moon, Hee Won; Yun, Yeo Min; Cho, Hyun Chan

2017-03-01

Cytomegalovirus (CMV) and Epstein-Barr virus (EBV) are increasingly important in immunocompromised patients. Nucleic acid extraction methods could affect the results of viral nucleic acid amplification tests. We compared two automated nucleic acid extraction systems for detecting CMV and EBV using real-time PCR assays. One hundred and fifty-three whole blood (WB) samples were tested for CMV detection, and 117 WB samples were tested for EBV detection. Viral nucleic acid was extracted in parallel by using QIAsymphony RGQ and QIAcube (Qiagen GmbH, Germany), and real-time PCR assays for CMV and EBV were performed with a Rotor-Gene Q real-time PCR cycler (Qiagen). Detection rates for CMV and EBV were compared, and agreements between the two systems were analyzed. The detection rate of CMV and EBV differed significantly between the QIAsymphony RGQ and QIAcube systems (CMV, 59.5% [91/153] vs 43.8% [67/153], P=0.0005; EBV, 59.0% [69/117] vs 42.7% [50/117], P=0.0008). The two systems showed moderate agreement for CMV and EBV detection (kappa=0.43 and 0.52, respectively). QIAsymphony RGQ showed a negligible correlation with QIAcube for quantitative EBV detection. QIAcube exhibited EBV PCR inhibition in 23.9% (28/117) of samples. Automated nucleic acid extraction systems have different performances and significantly affect the detection of viral pathogens. The QIAsymphony RGQ system appears to be superior to the QIAcube system for detecting CMV and EBV. A suitable sample preparation system should be considered for optimized nucleic acid amplification in clinical laboratories.

Diagnostic accuracy and applicability of a PCR system for the detection of Schistosoma mansoni DNA in human urine samples from an endemic area.

PubMed

Enk, Martin Johannes; Oliveira e Silva, Guilherme; Rodrigues, Nilton Barnabé

2012-01-01

Schistosomiasis caused by Schistosoma mansoni, one of the most neglected human parasitoses in Latin America and Africa, is routinely confirmed by microscopic visualization of eggs in stool. The main limitation of this diagnostic approach is its lack of sensitivity in detecting individual low worm burdens and consequently data on infection rates in low transmission settings are little reliable. According to the scientific literature, PCR assays are characterized by high sensitivity and specificity in detecting parasite DNA in biological samples. A simple and cost effective extraction method for DNA of Schistosoma mansoni from urine samples in combination with a conventional PCR assay was developed and applied in an endemic area. This urine based PCR system was tested for diagnostic accuracy among a population of a small village in an endemic area, comparing it to a reference test composed of three different parasitological techniques. The diagnostic parameters revealed a sensitivity of 100%, a specificity of 91.20%, positive and negative predictive values of 86.25% and 100%, respectively, and a test accuracy of 94.33%. Further statistical analysis showed a k index of 0.8806, indicating an excellent agreement between the reference test and the PCR system. Data obtained from the mouse model indicate the infection can be detected one week after cercariae penetration, opening a new perspective for early detection and patient management during this stage of the disease. The data indicate that this innovative PCR system provides a simple to handle and robust diagnostic tool for the detection of S. mansoni DNA from urine samples and a promising approach to overcome the diagnostic obstacles in low transmission settings. Furthermore the principals of this molecular technique, based on the examination of human urine samples may be useful for the diagnosis of other neglected tropical diseases that can be detected by trans-renal DNA.

Detection of BRAF-V600E and V600K in melanoma circulating tumour cells by droplet digital PCR.

PubMed

Reid, Anna L; Freeman, James B; Millward, Michael; Ziman, Melanie; Gray, Elin S

2015-10-01

Defining the BRAF mutation status in metastatic melanoma patients is critical to selecting patients for therapeutic treatment with targeted therapies. Circulating tumour cells (CTCs) can provide an alternative source of contemporaneous tumour genetic material. However methodologies to analyse the presence of rare mutations in a background of wild-type DNA requires a detailed assessment. Here we evaluate the sensitivity of two technologies for cancer mutation detection and the suitability of whole genome amplified DNA as a template for the detection of BRAF-V600 mutations. Serial dilutions of mutant BRAF-V600E DNA in wild-type DNA were tested using both competitive allele-specific PCR (castPCR) and droplet digital PCR (ddPCR), with and without previous whole genome amplification (WGA). Using immunomagnetic beads, we partially enriched CTCs from blood obtained from metastatic melanoma patients with confirmed BRAF mutation positive tumours and extracted RNA and DNA from the CTCs. We used RT-PCR of RNA to confirm the presence of melanoma cells in the CTC fraction then the DNAs of CTC positive fractions were WGA and tested for BRAF V600E or V600K mutations by ddPCRs. WGA DNA produced lower than expected fractional abundances by castPCR analysis but not by ddPCR. Moreover, ddPCR was found to be 200 times more sensitive than castPCR and in combination with WGA produced the most concordant results, with a limit of detection of 0.0005%. BRAF-V600E or V600K mutated DNA was detected in 77% and 44%, respectively, of enriched CTC fractions from metastatic melanoma patients carrying the corresponding mutations. Our results demonstrate that using ddPCR in combination with WGA DNA allows the detection with high sensitivity of cancer mutations in partially enriched CTC fractions. Copyright © 2014 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

Development and comparison of a real-time PCR assay for detection of Dichelobacter nodosus with culturing and conventional PCR: harmonisation between three laboratories

PubMed Central

2012-01-01

Background Ovine footrot is a contagious disease with worldwide occurrence in sheep. The main causative agent is the fastidious bacterium Dichelobacter nodosus. In Scandinavia, footrot was first diagnosed in Sweden in 2004 and later also in Norway and Denmark. Clinical examination of sheep feet is fundamental to diagnosis of footrot, but D. nodosus should also be detected to confirm the diagnosis. PCR-based detection using conventional PCR has been used at our institutes, but the method was laborious and there was a need for a faster, easier-to-interpret method. The aim of this study was to develop a TaqMan-based real-time PCR assay for detection of D. nodosus and to compare its performance with culturing and conventional PCR. Methods A D. nodosus-specific TaqMan based real-time PCR assay targeting the 16S rRNA gene was designed. The inclusivity and exclusivity (specificity) of the assay was tested using 55 bacterial and two fungal strains. To evaluate the sensitivity and harmonisation of results between different laboratories, aliquots of a single DNA preparation were analysed at three Scandinavian laboratories. The developed real-time PCR assay was compared to culturing by analysing 126 samples, and to a conventional PCR method by analysing 224 samples. A selection of PCR-products was cloned and sequenced in order to verify that they had been identified correctly. Results The developed assay had a detection limit of 3.9 fg of D. nodosus genomic DNA. This result was obtained at all three laboratories and corresponds to approximately three copies of the D. nodosus genome per reaction. The assay showed 100% inclusivity and 100% exclusivity for the strains tested. The real-time PCR assay found 54.8% more positive samples than by culturing and 8% more than conventional PCR. Conclusions The developed real-time PCR assay has good specificity and sensitivity for detection of D. nodosus, and the results are easy to interpret. The method is less time-consuming than either culturing or conventional PCR. PMID:22293440

Development, validation and testing costs of an in-house real-time PCR assay for the detection of Chlamydia trachomatis.

PubMed

Santos, Camila Gurgel Dos; Sabidó, Meritxell; Leturiondo, André Luiz; Ferreira, Cynthia de Oliveira; da Cruz, Thielle Pereira; Benzaken, Adele Schwartz

2017-03-01

To improve the screening of Chlamydia trachomatis(C. trachomatis) in Brazil, an accurate and affordable method is needed. The objective of this study was to develop and assess the performance and costs of a new in-house real-time PCR (qPCR) assay for the diagnosis of C. trachomatis infection. Asymptomatic women aged 14-25 years who attended primary health services in Manaus, Brazil, were screened for C. trachomatis using the Digene Hybrid Capture II CT-ID (HCII CT-ID) DNA test. A subset of cervical specimens were tested using an in-house qPCR and a commercial qPCR, ArtusC. trachomatis Plus RG PCR 96 CE (Artus qPCR) kit, as a reference test. A primer/probe based on the sequence of cryptic plasmid (CP) was designed. An economic evaluation was conducted from the provider's perspective. The primers were considered specific for C. trachomatis because they did not amplify any product from non-sexually transmitted bacterial species tested. Overall, 292 specimens were tested by both the commercial kit (Artus qPCR) and the in-house qPCR. Of those, one resulted in no amplification and was excluded from the analysis. The sensitivity, specificity, and positive and negative predictive values of the in-house qPCR were 99.5 % [95 % confidence interval (CI): 97.1-100], 95.1 % (95 % CI: 89-98.4), 97.4 % (95 % CI: 94-99.1) and 99.0 % (95 % CI: 94.5-100), respectively. The cost per case of C. trachomatis was £0.44 ($0.55) for HCII CT-ID, £1.16 ($1.45) for Artus qPCR and £1.06 ($1.33) for in-house qPCR. We have standardized an in-house qPCR to detect cervical C. trachomatis targeting CP. The in-house qPCR showed excellent accuracy and was more affordable than the commercial qPCR kit.

Detection of H5N1 high-pathogenicity avian influenza virus in meat and tracheal samples from experimentally infected chickens.

PubMed

Das, Amaresh; Spackman, Erica; Thomas, Colleen; Swayne, David E; Suarez, David L

2008-03-01

The Asian H5N1 highly pathogenic avian influenza (HPAI) virus causes a systemic disease with high mortality of poultry and is potentially zoonotic. In both chickens and ducks, the virus has been demonstrated to replicate in both cardiac and skeletal muscle cells. Experimentally, H5N1 HPAI virus has been transmitted to chickens through the consumption of raw infected meat. In this study, we investigated virus replication in cardiac and skeletal muscle and in the trachea of chickens after experimental intranasal inoculation with the H5N1 HPAI virus. The virus was detected in tissues by real-time reverse transcription-polymerase chain reaction (RRT-PCR) and virus isolation, and in the trachea by RRT-PCR and a commercial avian influenza (AI) viral antigen detection test. A modified RNA extraction protocol was developed for rapid detection of the virus in tissues by RRT-PCR. The H5N1 HPAI virus was sporadically detected in meat and the tracheas of infected birds without any clinical sign of disease as early as 6 hr postinfection (PI), and was detected in all samples tested at 24 hr PI and later. No differences in sensitivity were seen between virus isolation and RRT-PCR in meat samples. The AI viral antigen detection test on tracheal swabs was a useful method for identifying infected chickens when they were sick or dead, but was less sensitive in detecting infected birds when they were preclinical. This study provides data indicating that preslaughter tracheal swab testing can identify birds infected with HPAI among the daily mortality and prevent infected flocks from being sent to processing plants. In addition, the modified RNA extraction and RRT-PCR test on meat samples provide a rapid and sensitive method of identifying HPAI virus in illegal contraband or domestic meat samples.

Differential Detection of Enterovirus and Herpes Simplex Virus in Cerebrospinal Fluid by Real-Time RT-PCR.

PubMed

Sarquiz-Martínez, Brenda; González-Bonilla, César R; Santacruz-Tinoco, Clara Esperanza; Muñoz-Medina, José E; Pardavé-Alejandre, Héctor D; Barbosa-Cabrera, Elizabeth; Ramírez-González, José Ernesto; Díaz-Quiñonez, José Alberto

2017-01-01

Enterovirus (EV) and herpes simplex virus 1 and 2 (HSV1 and HSV2) are the main etiologic agents of central nervous system infections. Early laboratory confirmation of these infections is performed by viral culture of the cerebrospinal fluid (CSF), or the detection of specific antibodies in serum (e.g., HSV). The sensitivity of viral culture ranges from 65 to 75%, with a recovery time varying from 3 to 10 days. Serological tests are faster and easy to carry out, but they exhibit cross-reactivity between HSV1 and HSV2. Although molecular techniques are more sensitive (sensitivity >95%), they are more expensive and highly susceptible to cross-contamination. A real-time RT-PCR for the detection of EV, HSV1, and HSV2 was compared with end-point nested PCR. We tested 87 CSF samples of patients with a clinical diagnosis of viral meningitis or encephalitis. Fourteen samples were found to be positive by RT-PCR, but only 8 were positive by end-point PCR. The RT-PCR showed a specificity range of 94-100%, the negative predictive value was 100%, and the positive predictive value was 62, 100, and 28% for HSV1, HSV2, and EV, respectively. Real-time RT-PCR detected EV, HSV1, and HSV2 with a higher sensitivity and specificity than end-point nested RT-PCR. © 2017 S. Karger AG, Basel.

Development and validation of a multiplex real-time PCR method to simultaneously detect 47 targets for the identification of genetically modified organisms.

PubMed

Cottenet, Geoffrey; Blancpain, Carine; Sonnard, Véronique; Chuah, Poh Fong

2013-08-01

Considering the increase of the total cultivated land area dedicated to genetically modified organisms (GMO), the consumers' perception toward GMO and the need to comply with various local GMO legislations, efficient and accurate analytical methods are needed for their detection and identification. Considered as the gold standard for GMO analysis, the real-time polymerase chain reaction (RTi-PCR) technology was optimised to produce a high-throughput GMO screening method. Based on simultaneous 24 multiplex RTi-PCR running on a ready-to-use 384-well plate, this new procedure allows the detection and identification of 47 targets on seven samples in duplicate. To comply with GMO analytical quality requirements, a negative and a positive control were analysed in parallel. In addition, an internal positive control was also included in each reaction well for the detection of potential PCR inhibition. Tested on non-GM materials, on different GM events and on proficiency test samples, the method offered high specificity and sensitivity with an absolute limit of detection between 1 and 16 copies depending on the target. Easy to use, fast and cost efficient, this multiplex approach fits the purpose of GMO testing laboratories.

A new sensitive PCR assay for one-step detection of 12 IDH1/2 mutations in glioma.

PubMed

Catteau, Aurélie; Girardi, Hélène; Monville, Florence; Poggionovo, Cécile; Carpentier, Sabrina; Frayssinet, Véronique; Voss, Jesse; Jenkins, Robert; Boisselier, Blandine; Mokhtari, Karima; Sanson, Marc; Peyro-Saint-Paul, Hélène; Giannini, Caterina

2014-06-02

Mutations in isocitrate dehydrogenase genes IDH1 or IDH2 are frequent in glioma, and IDH mutation status is a strong diagnostic and prognostic marker. Current IDH mutation screening is performed with an immunohistochemistry (IHC) assay specific for IDH1 R132H, the most common mutation. Sequencing is recommended as a second-step test for IHC-negative or -equivocal cases. We developed and validated a new real-time quantitative polymerase chain reaction (PCR) assay for single-step detection of IDH1 R132H and 11 rare IDH1/2 mutations in formalin-fixed paraffin-embedded (FFPE) glioma samples. Performance of the IDH1/2 PCR assay was compared to IHC and Sanger sequencing. The IDH1/2 PCR assay combines PCR clamping for detection of 7 IDH1 and 5 IDH2 mutations, and Amplification Refractory Mutation System technology for specific identification of the 3 most common mutations (IDH1 R132H, IDH1 R132C, IDH2 R172K). Analytical sensitivity of the PCR assay for mutation detection was <5% for 11/12 mutations (mean: 3.3%), and sensitivity for mutation identification was very high (0.8% for IDH1 R132H; 1.2% for IDH1 R132C; 0.6% for IDH2 R172K). Assay performance was further validated on 171 clinical glioma FFPE samples; of these, 147 samples met the selection criteria and 146 DNA samples were successfully extracted. IDH1/2 status was successfully obtained in 91% of cases. All but one positive IDH1 R132H-IHC cases were concordantly detected by PCR and 3 were not detected by sequencing. Among the IHC-negative cases (n = 72), PCR detected 12 additional rare mutations (10 IDH1, 2 IDH2). All mutations detected by sequencing (n = 67) were concordantly detected by PCR and 5/66 sequencing-negative cases were PCR-positive (overall concordance: 96%). Analysis of synthetic samples representative of the 11 rare IDH1/2 mutations detected by the assay produced 100% correct results. The new IDH1/2 PCR assay has a high technical success rate and is more sensitive than Sanger sequencing. Positive concordance was 98% with IHC for IDH1 R132H detection and 100% with sequencing. The PCR assay can reliably be performed on FFPE samples and has a faster turnaround time than current IDH mutation detection algorithms. The assay should facilitate implementation of a comprehensive IDH1/2 testing protocol in routine clinical practice.

Abilities of the mCP Agar Method and CRENAME Alpha Toxin-Specific Real-Time PCR Assay To Detect Clostridium perfringens Spores in Drinking Water

PubMed Central

Maheux, Andrée F.; Bérubé, Ève; Boudreau, Dominique K.; Villéger, Romain; Cantin, Philippe; Boissinot, Maurice; Bissonnette, Luc

2013-01-01

We first determined the analytical specificity and ubiquity (i.e., the ability to detect all or most strains) of a Clostridium perfringens-specific real-time PCR (rtPCR) assay based on the cpa gene (cpa rtPCR) by using a bacterial strain panel composed of C. perfringens and non-C. perfringens Clostridium strains. All non-C. perfringens Clostridium strains tested negative, whereas all C. perfringens strains tested positive with the cpa rtPCR, for an analytical specificity and ubiquity of 100%. The cpa rtPCR assay was then used to confirm the identity of 116 putative C. perfringens isolates recovered after filtration of water samples and culture on mCP agar. Colonies presenting discordant results between the phenotype on mCP agar and cpa rtPCR were identified by sequencing the 16S rRNA and cpa genes. Four mCP−/rtPCR+ colonies were identified as C. perfringens, whereas 3 mCP+/rtPCR− colonies were identified as non-C. perfringens. The cpa rtPCR was negative with all 51 non-C. perfringens strains and positive with 64 of 65 C. perfringens strains. Finally, we compared mCP agar and a CRENAME (concentration and recovery of microbial particles, extraction of nucleic acids, and molecular enrichment) procedure plus cpa rtPCR (CRENAME + cpa rtPCR) for their abilities to detect C. perfringens spores in drinking water. CRENAME + cpa rtPCR detected as few as one C. perfringens CFU per 100 ml of drinking water sample in less than 5 h, whereas mCP agar took at least 25 h to deliver results. CRENAME + cpa rtPCR also allows the simultaneous and sensitive detection of Escherichia coli and C. perfringens from the same potable water sample. In itself, it could be used to assess the public health risk posed by drinking water potentially contaminated with pathogens more resistant to disinfection. PMID:24077714

Comparison of PCR and culture to the indirect fluorescent-antibody test for diagnosis of Potomac horse fever.

PubMed

Mott, J; Rikihisa, Y; Zhang, Y; Reed, S M; Yu, C Y

1997-09-01

Potomac horse fever is an acute systemic equine disease caused by Ehrlichia risticii. Currently, serologic methods are widely used to diagnose this disease. However, serologic methods cannot determine whether the horse is presently infected or has been exposed to ehrlichial antigens in the past. The purpose of the present study was to compare the sensitivities of the nested PCR and cell culture with that of the indirect fluorescent-antibody (IFA) test for the diagnosis of Potomac horse fever. Blood and fecal specimens serially collected from a pony experimentally infected with E. risticii Maryland, blood specimens serially collected from mice inoculated with E. risticii Ohio 380, and blood and/or fecal specimens collected from 27 horses which had clinical signs compatible with Potomac horse fever were examined. These horses resided in Kentucky, Indiana, Pennsylvania, and Vermont. The IFA test titer became positive after 6 days postinoculation (p.i.) for the pony. A culture of the blood of the pony was positive for E. risticii starting on day 1 and was positive through day 28 p.i. By the nested PCR, E. risticii was detectable in the blood and feces of the pony starting on day 1 and was detectable through day 32 p.i. E. risticii was detected in the blood of subclinically infected mice by the nested PCR. Twenty-two clinical specimens were seropositive for E. risticii by the IFA test, with titers ranging from 1:20 to 1:1,280. E. risticii was cultured from 95% (20 of 21) of seropositive clinical blood specimens. E. risticii was detected in the blood by PCR in 81% (17 of 20) of the culture-positive clinical specimens. The study indicated that the nested PCR is as sensitive as culture for detecting infection with E. risticii.

Single Fluorescence Channel-based Multiplex Detection of Avian Influenza Virus by Quantitative PCR with Intercalating Dye

PubMed Central

Ahberg, Christian D.; Manz, Andreas; Neuzil, Pavel

2015-01-01

Since its invention in 1985 the polymerase chain reaction (PCR) has become a well-established method for amplification and detection of segments of double-stranded DNA. Incorporation of fluorogenic probe or DNA intercalating dyes (such as SYBR Green) into the PCR mixture allowed real-time reaction monitoring and extraction of quantitative information (qPCR). Probes with different excitation spectra enable multiplex qPCR of several DNA segments using multi-channel optical detection systems. Here we show multiplex qPCR using an economical EvaGreen-based system with single optical channel detection. Previously reported non quantitative multiplex real-time PCR techniques based on intercalating dyes were conducted once the PCR is completed by performing melting curve analysis (MCA). The technique presented in this paper is both qualitative and quantitative as it provides information about the presence of multiple DNA strands as well as the number of starting copies in the tested sample. Besides important internal control, multiplex qPCR also allows detecting concentrations of more than one DNA strand within the same sample. Detection of the avian influenza virus H7N9 by PCR is a well established method. Multiplex qPCR greatly enhances its specificity as it is capable of distinguishing both haemagglutinin (HA) and neuraminidase (NA) genes as well as their ratio. PMID:26088868

On-chip quantitative detection of pathogen genes by autonomous microfluidic PCR platform.

PubMed

Tachibana, Hiroaki; Saito, Masato; Shibuya, Shogo; Tsuji, Koji; Miyagawa, Nobuyuki; Yamanaka, Keiichiro; Tamiya, Eiichi

2015-12-15

Polymerase chain reaction (PCR)-based genetic testing has become a routine part of clinical diagnoses and food testing. In these fields, rapid, easy-to-use, and cost-efficient PCR chips are expected to be appeared for providing such testing on-site. In this study, a new autonomous disposable plastic microfluidic PCR chip was created, and was utilized for quantitative detection of pathogenic microorganisms. To control the capillary flow of the following solution in the PCR microchannel, a driving microchannel was newly designed behind the PCR microchannel. This allowed the effective PCR by simply dropping the PCR solution onto the inlet without any external pumps. In order to achieve disposability, injection-molded cyclo-olefin polymer (COP) of a cost-competitive plastic was used for the PCR chip. We discovered that coating the microchannel walls with non-ionic surfactant produced a suitable hydrophilic surface for driving the capillary flow through the 1250-mm long microchannel. As a result, quantitative real-time PCR with the lowest initial concentration of human, Escherichia coli (E. coli), and pathogenic E. coli O157 genomic DNA of 4, 0.0019, 0.031 pg/μl, respectively, was successfully achieved in less than 18 min. Our results indicate that the platform presented in this study provided a rapid, easy-to-use, and low-cost real-time PCR system that could be potentially used for on-site gene testing. Copyright © 2015 Elsevier B.V. All rights reserved.

Development and application of two independent real-time PCR assays to detect clinically relevant Mucorales species.

PubMed

Springer, Jan; Goldenberger, Daniel; Schmidt, Friderike; Weisser, Maja; Wehrle-Wieland, Elisabeth; Einsele, Hermann; Frei, Reno; Löffler, Jürgen

2016-03-01

PCR-based detection of Mucorales species could improve diagnosis of suspected invasive fungal infection, leading to a better patient outcome. This study describes two independent probe-based real-time PCR tests for detection of clinically relevant Mucorales, targeting specific fragments of the 18S and the 28S rRNA genes. Both assays have a short turnaround time, allow fast, specific and very sensitive detection of clinically relevant Mucorales and have the potential to be used as quantitative tests. They were validated on various clinical samples (fresh and formalin-fixed paraffin-embedded specimens, mainly biopsies, n = 17). The assays should be used as add-on tools to complement standard techniques; a combined approach of both real-time PCR assays has 100 % sensitivity. Genus identification by subsequent sequencing is possible for amplicons of the 18S PCR assay. In conclusion, combination of the two independent Mucorales assays described in this study, 18S and 28S, detected all clinical samples associated with proven Mucorales infection (n = 10). Reliable and specific identification of Mucorales is a prerequisite for successful antifungal therapy as these fungi show intrinsic resistance to voriconazole and caspofungin.

Detection of K1 antigen of Escherichia coli rods isolated from pregnant women and neonates.

PubMed

Kaczmarek, Agnieszka; Budzyńska, Anna; Gospodarek, Eugenia

2014-09-01

The K1 antigen is an important virulence determinant of Escherichia coli strains and has been shown to be associated particularly with neonatal meningitis, bacteraemia and septicaemia. Thus, its detection seems to be useful, especially in the case of E. coli strains isolated from pregnant women and newborns. In this study, the sensitivity and specificity of the latex agglutination test (Pastorex Meningitis) for identification of E. coli serogroup K1 were assessed, using PCR as the gold standard. Our results showed that consistency of results between latex agglutination test and PCR amounted to 98.5%. Therefore, Pastorex Meningitis is a good alternative to PCR and could be used for rapid K1 antigen detection, especially in local non-specialized laboratories with limited resources where PCR assay is not applied.

A one-step multiplex RT-PCR assay for simultaneous detection of four viruses that infect peach.

PubMed

Yu, Y; Zhao, Z; Jiang, D; Wu, Z; Li, S

2013-10-01

A multiplex reverse transcription polymerase chain reaction (mRT-PCR) assay was developed to enable the simultaneous detection and differentiation of four viruses that infect peach, namely Apple chlorotic leaf spot virus (ACLSV), Cherry green ring mottle virus (CGRMV), Prunus necrotic ringspot virus (PNRSV) and Apricot pseudo-chlorotic leaf spot virus (APCLSV). In this study, four pairs of primers, one specific for each virus, were designed; the corresponding PCR products were 632, 439, 346 and 282 bp in length for ACLSV, CGRMV, PNRSV and APCLSV, respectively, and the fragments could be distinguished clearly by agarose gel electrophoresis. The sensitivity and specificity of the method were tested using individual RT-PCR and enzyme-linked immunosorbent assay (ELISA), and the identity of the RT-PCR amplification products was also confirmed by DNA sequencing. The results of RT-PCR and ELISA, along with batch detection using samples collected from peach orchards, revealed that this rapid and simple technique is an effective way to identify the four viruses simultaneously. The mRT-PCR assay described in this study was developed for the simultaneous detection of four peach viruses from infected peach samples is reliable and sensitive. In contrast to conventional uniplex RT-PCR, mRT-PCR is more efficient, reducing costs, time and handling when testing large numbers of samples. This rapid and simple method is useful for large-scale surveys of viruses that infect peach. © 2013 The Society for Applied Microbiology.

Screening for Chlamydia trachomatis in Egyptian women with unexplained infertility, comparing real-time PCR techniques to standard serology tests: case control study.

PubMed

Abdella, Rana M A; Abdelmoaty, Hatem I; Elsherif, Rasha H; Sayed, Ahmed Mahmoud; Sherif, Nadine Alaa; Gouda, Hisham M; El Lithy, Ahmed; Almohamady, Maged; Abdelbar, Mostafa; Hosni, Ahmed Naguib; Magdy, Ahmed; Ma, Youssef

2015-06-02

To study the prevalence of Chlamydia infection in women with primary and secondary unexplained infertility using ELISA technique for antibody detection and real time, fully automated PCR for antigen detection and to explore its association with circulating antisperm antibodies (ASA). A total of 50 women with unexplained infertility enrolled in this case control study and a control group of 44 infertile women with a known cause of infertility. Endocervical specimens were collected for Chlamydia antigen detection using PCR and serum samples for antibodies detection. Circulating anti-sperm antibodies were detected using sperm antibody Latex Agglutination tests. The overall prevalence of Chlamydial infection in unexplained infertility cases as detected by both ELISA and PCR was 40 % (20/50). The prevalence of current Chlamydial genital infection as detected by real-time PCR was only 6.0 % (3/50); two of which were also IgM positive. Prevalence of ASA was 6.0 % (3/50); all were sero-negative for anti-C.trachomatis IgM and were PCR negative. The incidence of Chlamydial infection in Egyptian patients with unexplained infertility is relatively high. In the setting of fertility investigations; screening for anti. C.trachomatis antibodies using ELISA, and treatment of positive cases should be considered. The presence of circulating ASA does not correlate with the presence of old or current Chlamydia infection in women with unexplained infertility.

Concurrent infections of pseudorabies virus and porcine bocavirus in China detected by duplex nanoPCR.

PubMed

Luo, Yakun; Liang, Lin; Zhou, Ling; Zhao, Kai; Cui, Shangjin

2015-07-01

Nanoparticle-assisted polymerase chain reaction (nanoPCR) is a novel method for the simple, rapid, and specific amplification of DNA and has been used to detect viruses. A duplex nanoPCR molecular detection system was developed to detect pseudorabies virus (PRV) and porcine bocavirus (PBoV). Primers were selected to target conserved regions within the PRV gE gene and the PBoV NS1 gene. Under optimized nanoPCR reaction conditions, two specific fragments of 316 bp (PRV) and 996 bp (PBoV) were amplified by the duplex nanoPCR with a detection limit of 6 copies for PRV and 95 copies for PBoV; no fragments were amplified when other porcine viruses were used as template. When used to test 550 clinical samples, the duplex nanoPRC assay and a conventional duplex PCR assay provided very similar results (98.1% consistency); single PRV infections, single PBoV infections, and concurrent PRV and PBoV infections were detected in 37%, 15%, and 9% of the samples, respectively. The results indicate that the novel duplex nanoPCR assay is useful for the rapid detection of PRV and PBoV in pigs. Copyright © 2015 Elsevier B.V. All rights reserved.

Comparison of molecular tests for the diagnosis of malaria in Honduras

PubMed Central

2012-01-01

Background Honduras is a tropical country with more than 70% of its population living at risk of being infected with either Plasmodium vivax or Plasmodium falciparum. Laboratory diagnosis is a very important factor for adequate treatment and management of malaria. In Honduras, malaria is diagnosed by both, microscopy and rapid diagnostic tests and to date, no molecular methods have been implemented for routine diagnosis. However, since mixed infections, and asymptomatic and low-parasitaemic cases are difficult to detect by light microscopy alone, identifying appropriate molecular tools for diagnostic applications in Honduras deserves further study. The present study investigated the utility of different molecular tests for the diagnosis of malaria in Honduras. Methods A total of 138 blood samples collected as part of a clinical trial to assess the efficacy of chloroquine were used: 69 microscopically confirmed P. falciparum positive samples obtained on the day of enrolment and 69 follow-up samples obtained 28 days after chloroquine treatment and shown to be malaria negative by microscopy. Sensitivity and specificity of microscopy was compared to an 18 s ribosomal RNA gene-based nested PCR, two single-PCR reactions designed to detect Plasmodium falciparum infections, one single-PCR to detect Plasmodium vivax infections, and one multiplex one-step PCR reaction to detect both parasite species. Results Of the 69 microscopically positive P. falciparum samples, 68 were confirmed to be P. falciparum-positive by two of the molecular tests used. The one sample not detected as P. falciparum by any of the molecular tests was shown to be P. vivax-positive by a reference molecular test indicating a misdiagnosis by microscopy. The reference molecular test detected five cases of P. vivax/P. falciparum mixed infections, which were not recognized by microscopy as mixed infections. Only two of these mixed infections were recognized by a multiplex test while a P. vivax-specific polymerase chain reaction (PCR) detected three of them. In addition, one of the day 28 samples, previously determined to be malaria negative by microscopy, was shown to be P. vivax-positive by three of the molecular tests specific for this parasite. Conclusions Molecular tests are valuable tools for the confirmation of Plasmodium species and in detecting mixed infections in malaria endemic regions. PMID:22513192

Comparison of molecular tests for the diagnosis of malaria in Honduras.

PubMed

Fontecha, Gustavo A; Mendoza, Meisy; Banegas, Engels; Poorak, Mitra; De Oliveira, Alexandre M; Mancero, Tamara; Udhayakumar, Venkatachalam; Lucchi, Naomi W; Mejia, Rosa E

2012-04-18

Honduras is a tropical country with more than 70% of its population living at risk of being infected with either Plasmodium vivax or Plasmodium falciparum. Laboratory diagnosis is a very important factor for adequate treatment and management of malaria. In Honduras, malaria is diagnosed by both, microscopy and rapid diagnostic tests and to date, no molecular methods have been implemented for routine diagnosis. However, since mixed infections, and asymptomatic and low-parasitaemic cases are difficult to detect by light microscopy alone, identifying appropriate molecular tools for diagnostic applications in Honduras deserves further study. The present study investigated the utility of different molecular tests for the diagnosis of malaria in Honduras. A total of 138 blood samples collected as part of a clinical trial to assess the efficacy of chloroquine were used: 69 microscopically confirmed P. falciparum positive samples obtained on the day of enrollment and 69 follow-up samples obtained 28 days after chloroquine treatment and shown to be malaria negative by microscopy. Sensitivity and specificity of microscopy was compared to an 18 s ribosomal RNA gene-based nested PCR, two single-PCR reactions designed to detect Plasmodium falciparum infections, one single-PCR to detect Plasmodium vivax infections, and one multiplex one-step PCR reaction to detect both parasite species. Of the 69 microscopically positive P. falciparum samples, 68 were confirmed to be P. falciparum-positive by two of the molecular tests used. The one sample not detected as P. falciparum by any of the molecular tests was shown to be P. vivax-positive by a reference molecular test indicating a misdiagnosis by microscopy. The reference molecular test detected five cases of P. vivax/P. falciparum mixed infections, which were not recognized by microscopy as mixed infections. Only two of these mixed infections were recognized by a multiplex test while a P. vivax-specific polymerase chain reaction (PCR) detected three of them. In addition, one of the day 28 samples, previously determined to be malaria negative by microscopy, was shown to be P. vivax-positive by three of the molecular tests specific for this parasite. Molecular tests are valuable tools for the confirmation of Plasmodium species and in detecting mixed infections in malaria endemic regions.

A Cytolethal Distending Toxin Gene-Based Multiplex PCR Assay for Campylobacter jejuni, C. fetus, C. coli, C. upsaliensis, C. hyointestinalis, and C. lari.

PubMed

Kamei, Kazumasa; Kawabata, Hiroki; Asakura, Masahiro; Samosornsuk, Worada; Hinenoya, Atsushi; Nakagawa, Shinsaku; Yamasaki, Shinji

2016-05-20

In this study, we devised a multiplex PCR assay based on the gene of cytolethal distending toxin (cdt) B subunit to simultaneously detect and discriminate Campylobacter jejuni, C. fetus, C. coli, C. upsaliensis, C. hyointestinalis, and C. lari. Species-specific PCR products were successfully obtained from all 38 C. jejuni, 12 C. fetus, 39 C. coli, 22 C. upsaliensis, 24 C. hyointestinalis, and 7 C. lari strains tested. On the other hand, no specific PCR products were obtained from other campylobacters and bacterial species tested (41 strains in total). The proposed multiplex PCR assay is a valuable tool for detection and descrimination of 6 major Campylobacter species, that are associated with gastrointestinal diseases in humans.

Toxoplasma gondii infections in chickens - performance of various antibody detection techniques in serum and meat juice relative to bioassay and DNA detection methods.

PubMed

Schares, G; Koethe, M; Bangoura, B; Geuthner, A-C; Randau, F; Ludewig, M; Maksimov, P; Sens, M; Bärwald, A; Conraths, F J; Villena, I; Aubert, D; Opsteegh, M; Van der Giessen, J

2018-05-19

Chickens, especially if free-range, are frequently exposed to Toxoplasma gondii, and may represent an important reservoir for T. gondii. Poultry products may pose a risk to humans, when consumed undercooked. In addition, chickens are regarded as sensitive indicators for environmental contamination with T. gondii oocysts and have been used as sentinels. The aim of the present study was to determine the suitability of commonly used antibody detection methods, i.e. the modified agglutination test (MAT), IFAT and ELISA to detect T. gondii-infected chickens. Samples of experimentally and naturally infected chickens were used. The infection state of all chickens was determined by Magnetic-Capture (MC-) real-time PCR (RT PCR). Naturally exposed chickens were additionally examined by mouse bioassay and conventional RT PCR on acidic pepsin digests (PD-RT PCR). Blood serum and meat juice of various sources were tested for antibodies to T. gondii. In naturally infected chickens, there was substantial agreement between the mouse bioassay and MC-RT PCR or the mouse bioassay and conventional PD-RT PCR. PD-RT PCR was slightly more sensitive than MC-RT PCR, as all (26/26) bioassay-positive chickens also tested positive in at least one of the tissues tested (heart, drumstick). By MC-RT PCR, 92.3% (24/26) of the naturally infected bioassay-positive chickens were positive. The diagnostic sensitivity of MC-RT PCR was clearly related to the organ examined. Based on a quantitative assessment of the MC-RT PCR results in experimentally infected chickens, brain and heart tissues harbored an at least 100 times higher parasite concentration than breast, thigh or drumstick musculature. In naturally infected chickens, only three out of 24 birds, which were MC-RT PCR-positive in heart samples, also tested positive in drumstick musculature. Under experimental conditions, the agreement between MC-RT PCR and the serological techniques revealed 100% diagnostic sensitivity and specificity. Under field conditions, examinations of sera by ELISA, IFAT and MAT showed good performance in identifying chickens that were positive in either a mouse bioassay, MC-RT PCR, or PD-RT PCR as illustrated by diagnostic sensitivities of 87.5%, 87.5% and 65.2%, respectively, and diagnostic specificities of 86.2%, 82.8% and 100%, respectively. The examination of meat juice samples from breast, drumstick or heart musculature revealed similar or even better results in the ELISA. The results in the MAT with meat juice from breast musculature were less consistent than those of ELISA and IFAT because a number of negative chickens tested false-positive in the MAT. The MAT performed similar to ELISA and IFAT when applied to test meat juice samples collected from heart, thigh or drumstick musculature. Copyright © 2018 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.

Real-time PCR for rapidly detecting aniline-degrading bacteria in activated sludge.

PubMed

Kayashima, Takakazu; Suzuki, Hisako; Maeda, Toshinari; Ogawa, Hiroaki I

2013-05-01

We developed a detection method that uses quantitative real-time PCR (qPCR) and the TaqMan system to easily and rapidly assess the population of aniline-degrading bacteria in activated sludge prior to conducting a biodegradability test on a chemical compound. A primer and probe set for qPCR was designed by a multiple alignment of conserved amino acid sequences encoding the large (α) subunit of aniline dioxygenase. PCR amplification tests showed that the designed primer and probe set targeted aniline-degrading strains such as Acidovorax sp., Gordonia sp., Rhodococcus sp., and Pseudomonas putida, thereby suggesting that the developed method can detect a wide variety of aniline-degrading bacteria. There was a strong correlation between the relative copy number of the α-aniline dioxygenase gene in activated sludge obtained with the developed qPCR method and the number of aniline-degrading bacteria measured by the Most Probable Number method, which is the conventional method, and a good correlation with the lag time of the BOD curve for aniline degradation produced by the biodegradability test in activated sludge samples collected from eight different wastewater treatment plants in Japan. The developed method will be valuable for the rapid and accurate evaluation of the activity of inocula prior to conducting a ready biodegradability test. Copyright © 2013 Elsevier Ltd. All rights reserved.

A Comparative Analysis of Polymerase Chain Reaction and Direct Fluorescent Antibody Test for Diagnosis of Genital Herpes.

PubMed

Patwardhan, Vrushali; Bhalla, Preena; Rawat, Deepti; Garg, Vijay Kumar; Sardana, Kabir; Sethi, Sumit

2017-01-01

To compare laboratory tests that can simultaneously detect and type herpes simplex virus (HSV) directly from the genital ulcer specimens in clinically suspected cases of genital herpes. A study was conducted over 10 months and 44 adult male and female patients clinically suspected with genital herpes were recruited. Genital ulcer swab specimens were subjected to glycoprotein-G gene-based conventional polymerase chain reaction (PCR) and commercially available direct fluorescent antibody (DFA) test and the results were compared. PCR for HSV was positive in 82% (36/44) cases. DFA was positive in 68.2% (30/44) cases. There was 100% agreement between HSV types detected by DFA and PCR. The strength of agreement between the results was better in primary genital herpes than recurrent cases. PCR was found to be better in the detection of HSV in recurrent genital herpes patients. It is a better modality, especially when genital herpes clinically presents with ulcerative or crusted lesions, and is also a cheaper alternative as compared to DFA.

Detection of Echinococcus multilocularis by MC-PCR: evaluation of diagnostic sensitivity and specificity without gold standard.

PubMed

Wahlström, Helene; Comin, Arianna; Isaksson, Mats; Deplazes, Peter

2016-01-01

A semi-automated magnetic capture probe-based DNA extraction and real-time PCR method (MC-PCR), allowing for a more efficient large-scale surveillance of Echinococcus multilocularis occurrence, has been developed. The test sensitivity has previously been evaluated using the sedimentation and counting technique (SCT) as a gold standard. However, as the sensitivity of the SCT is not 1, test characteristics of the MC-PCR was also evaluated using latent class analysis, a methodology not requiring a gold standard. Test results, MC-PCR and SCT, from a previous evaluation of the MC-PCR using 177 foxes shot in the spring (n=108) and autumn 2012 (n=69) in high prevalence areas in Switzerland were used. Latent class analysis was used to estimate the test characteristics of the MC-PCR. Although it is not the primary aim of this study, estimates of the test characteristics of the SCT were also obtained. This study showed that the sensitivity of the MC-PCR was 0.88 [95% posterior credible interval (PCI) 0.80-0.93], which was not significantly different than the SCT, 0.83 (95% PCI 0.76-0.88), which is currently considered as the gold standard. The specificity of both tests was high, 0.98 (95% PCI 0.94-0.99) for the MC-PCR and 0.99 (95% PCI 0.99-1) for the SCT. In a previous study, using fox scats from a low prevalence area, the specificity of the MC-PCR was higher, 0.999% (95% PCI 0.997-1). One reason for the lower estimate of the specificity in this study could be that the MC-PCR detects DNA from infected but non-infectious rodents eaten by foxes. When using MC-PCR in low prevalence areas or areas free from the parasite, a positive result in the MC-PCR should be regarded as a true positive. The sensitivity of the MC-PCR (0.88) was comparable to the sensitivity of SCT (0.83).

A Novel Pan-Flavivirus Detection and Identification Assay Based on RT-qPCR and Microarray

PubMed Central

Sachse, Konrad; Ziegler, Ute; Keller, Markus

2017-01-01

The genus Flavivirus includes arthropod-borne viruses responsible for a large number of infections in humans and economically important animals. While RT-PCR protocols for specific detection of most Flavivirus species are available, there has been also a demand for a broad-range Flavivirus assay covering all members of the genus. It is particularly challenging to balance specificity at genus level with equal sensitivity towards each target species. In the present study, a novel assay combining a SYBR Green-based RT-qPCR with a low-density DNA microarray has been developed. Validation experiments confirmed that the RT-qPCR exhibited roughly equal sensitivity of detection and quantification for all flaviviruses tested. These PCR products are subjected to hybridization on a microarray carrying 84 different oligonucleotide probes that represent all known Flavivirus species. This assay has been used as a screening and confirmation tool for Flavivirus presence in laboratory and field samples, and it performed successfully in international External Quality Assessment of NAT studies. Twenty-six Flavivirus strains were tested with the assay, showing equivalent or superior characteristics compared with the original or even with species-specific RT-PCRs. As an example, test results on West Nile virus detection in a panel of 340 mosquito pool samples from Greece are presented. PMID:28626758

Detection of Japanese Encephalitis Virus RNA in Human Throat Samples in Laos - A Pilot study.

PubMed

Bharucha, Tehmina; Sengvilaipaseuth, Onanong; Seephonelee, Malee; Vongsouvath, Malavanh; Vongsouvath, Manivanh; Rattanavong, Sayaphet; Piorkowski, Géraldine; Lecuit, Marc; Gorman, Christopher; Pommier, Jean-David; Newton, Paul N; de Lamballerie, Xavier; Dubot-Pérès, Audrey

2018-05-22

Japanese encephalitis virus (JEV) is the most commonly identified cause of acute encephalitis syndrome (AES) in Asia. The WHO recommended test is anti-JEV IgM-antibody-capture-enzyme-linked-immunosorbent-assay (JEV MAC-ELISA). However, data suggest this has low positive predictive value, with false positives related to other Flavivirus infections and vaccination. JEV RT-PCR in cerebrospinal fluid (CSF) and/or serum is highly specific, but is rarely positive; 0-25% of patients that fulfil the WHO definition of JE (clinical Acute Encephalitis Syndrome (AES) and JEV MAC-ELISA positive). Testing other body fluids by JEV RT-qPCR may improve the diagnosis. As a pilot study thirty patients admitted to Mahosot Hospital 2014-2017, recruited to the South-East-Asia-Encephalitis study, were tested by JEV MAC-ELISA and two JEV real-time RT-PCR (RT-qPCR) assays (NS2A and NS3). Eleven (36.7%) were JEV MAC-ELISA positive. Available CSF and serum samples of these patients were JEV RT-qPCR negative but 2 (7%) had JEV RNA detected in their throat swabs. JEV RNA was confirmed by re-testing, and sequencing of RT-qPCR products. As the first apparent report of JEV RNA detection in human throat samples, the provides new perspectives on human JEV infection, potentially informing improving JEV detection. We suggest that testing patients' throat swabs for JEV RNA is performed, in combination with molecular and serological CSF and serum investigations, on a larger scale to investigate the epidemiology of the presence of JEV in human throats. Throat swabs are an easy and non-invasive tool that could be rolled out to a wider population to improve knowledge of JEV molecular epidemiology.

Rapid and Accurate Diagnosis of Acute Pyogenic Meningitis Due to Streptococcus Pneumoniae, Haemophilus influenzae Type b and Neisseria meningitidis Using A Multiplex PCR Assay.

PubMed

Seth, Rajeev; Murthy, Peela Sree Ramchandra; Sistla, Sujatha; Subramanian, Mahadevan; Tamilarasu, Kadhiravan

2017-09-01

Acute bacterial meningitis is one of the major causes of morbidity and mortality in children and geriatric population, especially in developing countries. Methods of identification are standard culture and other phenotypic tests in many resource poor settings. To use molecular methods for the improvement of aetiological diagnosis of acute pyogenic meningitis in patients. CSF samples of 125 patients were included for the study. Gram staining and culture were performed according to standard procedures. Antigen was detected using commercial latex agglutination test kit. Multiplex PCR was performed using previously published primers and protocols. Fischer's exact test was used for finding association between presence of the disease and clinical/biochemical parameters, considering two tailed p<0.05 as statistically significant. Sensitivity, specificity, positive and negative predictive values were calculated using Graphpad QuicCalc software. A total of 39 cases (31.2%) were confirmed to be of acute pyogenic meningitis based on biochemical methods. Only 10/39 was positive for the three organisms tested. Multiplex PCR was able to detect one additional isolate each of Streptococcus pneumoniae and Haemophilus influenzae type b. When compared with multiplex PCR as the gold standard, culture and latex agglutination tests had same sensitivity (80%), specificity (100%), PPV (100%) and NPV (97.8%), whereas Gram stain had poor sensitivity (40%) and good specificity (95.6%). Detection rates were higher in multiplex PCR for the two organisms Streptococcus pneumoniae and Haemophilus influenzae type b. Multiplex PCR was more sensitive than culture or antigen detection, and employing this assay can significantly increase the speed and accuracy of identification of the pathogen.

Perirectal Swab Surveillance for Clostridium difficile by Use of Selective Broth Preamplification and Real-Time PCR Detection of tcdB ▿

PubMed Central

Curry, Scott R.; Schlackman, Jessica L.; Hamilton, Travis M.; Henderson, Tatianna K.; Brown, Nakita T.; Marsh, Jane W.; Shutt, Kathleen A.; Brooks, Maria M.; Pasculle, A. William; Muto, Carlene A.; Harrison, Lee H.

2011-01-01

Active surveillance testing to identify and isolate asymptomatic carriers of toxigenic Clostridium difficile has been limited by the lack of a test that is sensitive, specific, and timely enough to serve as an infection control tool. We tested DNA preamplified from perirectal surveillance specimens in a liquid medium selective for C. difficile by using a modified commercial real-time PCR assay. All fermenting specimens were subcultured, and isolates were tested for toxigenicity. Culture-positive toxigenic isolates served as the gold standard for comparison with the broth preamplification/PCR assay. The limit of detection for the assay was 1 CFU. Relative to toxigenic anaerobic culture, the sensitivity, specificity, and positive and negative predictive values of this assay were 70/70 (100.0%), 422/426 (99.1%), 70/74 (94.6%), and 422/422 (100.0%), respectively. These data demonstrate that selective broth preamplification and real-time PCR of perirectal swab specimens constitute a practical approach to the detection of asymptomatic C. difficile carriage. PMID:21880961

Next-Generation Genotyping by Digital PCR to Detect and Quantify the BRAF V600E Mutation in Melanoma Biopsies.

PubMed

Lamy, Pierre-Jean; Castan, Florence; Lozano, Nicolas; Montélion, Cécile; Audran, Patricia; Bibeau, Frédéric; Roques, Sylvie; Montels, Frédéric; Laberenne, Anne-Claire

2015-07-01

The detection of the BRAF V600E mutation in melanoma samples is used to select patients who should respond to BRAF inhibitors. Different techniques are routinely used to determine BRAF status in clinical samples. However, low tumor cellularity and tumor heterogeneity can affect the sensitivity of somatic mutation detection. Digital PCR (dPCR) is a next-generation genotyping method that clonally amplifies nucleic acids and allows the detection and quantification of rare mutations. Our aim was to evaluate the clinical routine performance of a new dPCR-based test to detect and quantify BRAF mutation load in 47 paraffin-embedded cutaneous melanoma biopsies. We compared the results obtained by dPCR with high-resolution melting curve analysis and pyrosequencing or with one of the allele-specific PCR methods available on the market. dPCR showed the lowest limit of detection. dPCR and allele-specific amplification detected the highest number of mutated samples. For the BRAF mutation load quantification both dPCR and pyrosequencing gave similar results with strong disparities in allele frequencies in the 47 tumor samples under study (from 0.7% to 79% of BRAF V600E mutations/sample). In conclusion, the four methods showed a high degree of concordance. dPCR was the more-sensitive method to reliably and easily detect mutations. Both pyrosequencing and dPCR could quantify the mutation load in heterogeneous tumor samples. Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

Protocol for the use of a rapid real-time PCR method for the detection of HIV-1 proviral DNA using double-stranded primer.

PubMed

Pau, Chou-Pong; Wells, Susan K; Granade, Timothy C

2012-01-01

This chapter describes a real-time PCR method for the detection of HIV-1 proviral DNA in whole blood samples using a novel double-stranded primer system. The assay utilizes a simple commercially available DNA extraction method and a rapid and easy-to-perform real-time PCR protocol to consistently detect a minimum of four copies of HIV-1 group M proviral DNA in as little as 90 min after sample (whole blood) collection. Co-amplification of the human RNase P gene serves as an internal control to monitor the efficiency of both the DNA extraction and amplification. Once the assay is validated properly, it may be suitable as an alternative confirmation test for HIV-1 infections in a variety of HIV testing venues including the mother-to-child transmission testing sites, clinics, and diagnostic testing centers.

A rapid single-tube protocol for HAV detection by nested real-time PCR.

PubMed

Hu, Yuan; Arsov, Ivica

2014-09-01

Infections by food-borne viruses such as hepatitis A virus (HAV) and norovirus are significant public health concerns worldwide. Since food-borne viruses are rarely confirmed through direct isolation from contaminated samples, highly sensitive molecular techniques remain the methods of choice for the detection of viral genetic material. Our group has previously developed a specific nested real-time PCR (NRT-PCR) assay for HAV detection that improved overall sensitivity. Furthermore in this study, we have developed a single-tube NRT-PCR approach for HAV detection in food samples that reduces the likelihood of cross contamination between tubes during sample manipulation. HAV RNA was isolated from HAV-spiked food samples and HAV-infected cell cultures. All reactions following HAV RNA isolation, including conventional reverse transcriptase PCR, nested-PCR, and RT-PCR were performed in a single tube. Our results demonstrated that all the samples tested positive by RT-PCR and nested-PCR were also positive by a single-tube NRT-PCR. The detection limits observed for HAV-infected cell cultures and HAV-spiked green onions were 0.1 and 1 PFU, respectively. This novel method retained the specificity and robustness of the original NRT-PCR method, while greatly reducing sample manipulation, turnaround time, and the risk of carry-over contamination. Single-tube NRT-PCR thus represents a promising new tool that can potentially facilitate the detection of HAV in foods thereby improving food safety and public health.

An Epidemiologic Analysis of Chlamydia trachomatis and Neisseria gonorrhoeae Infections in Female Federal Prisoners

DTIC Science & Technology

2002-01-01

compared to culture.70,72 Use of PCR to detect GC has yielded sensitivities between 92-100% and specificities ranging from 96-99% on endocervical...specimen.73,74 With use of urine specimens, PCR has yielded sensitivities ranging from 65 to 92% and 96-99% respectively, for GC detection .74...21 An important limitation of PCR and LCR is that the tests may detect CT or GC nucleic acid remaining after therapy has been administered

[Sensitivity and specificity of nested PCR pyrosequencing in hepatitis B virus drug resistance gene testing].

PubMed

Sun, Shumei; Zhou, Hao; Zhou, Bin; Hu, Ziyou; Hou, Jinlin; Sun, Jian

2012-05-01

To evaluate the sensitivity and specificity of nested PCR combined with pyrosequencing in the detection of HBV drug-resistance gene. RtM204I (ATT) mutant and rtM204 (ATG) nonmutant plasmids mixed at different ratios were detected for mutations using nested-PCR combined with pyrosequencing, and the results were compared with those by conventional PCR pyrosequencing to analyze the linearity and consistency of the two methods. Clinical specimens with different viral loads were examined for drug-resistant mutations using nested PCR pyrosequencing and nested PCR combined with dideoxy sequencing (Sanger) for comparison of the detection sensitivity and specificity. The fitting curves demonstrated good linearity of both conventional PCR pyrosequencing and nested PCR pyrosequencing (R(2)>0.99, P<0.05). Nested PCR showed a better consistency with the predicted value than conventional PCR, and was superior to conventional PCR for detection of samples containing 90% mutant plasmid. In the detection of clinical specimens, Sanger sequencing had a significantly lower sensitivity than nested PCR pyrosequencing (92% vs 100%, P<0.01). The detection sensitivity of Sanger sequencing varied with the viral loads, especially in samples with low viral copies (HBV DNA ≤3log10 copies/ml), where the sensitivity was 78%, significantly lower than that of pyrosequencing (100%, P<0.01). Neither of the two methods yielded positive results for the negative control samples, suggesting their good specificity. Compared with nested PCR and Sanger sequencing method, nested PCR pyrosequencing has a higher sensitivity especially in clinical specimens with low viral copies, which can be important for early detection of HBV mutant strains and hence more effective clinical management.

AccuCopy quantification combined with pre-amplification of long-distance PCR for fast analysis of intron 22 inversion in haemophilia A.

PubMed

Ding, Qianlan; Wu, Xi; Lu, Yeling; Chen, Changming; Shen, Rui; Zhang, Xi; Jiang, Zhengwen; Wang, Xuefeng

2016-07-01

To develop a digitalized intron 22 inversion (Inv22) detection in patients with severe haemophilia A. The design included two tests: A genotyping test included two multiplex pre-amplification of LD-PCR (PLP) with two combinations of five primers to amplify wild-type and chimeric int22h alleles; a carrier mosaicism test was similar to the genotyping test except only amplification of chimeric int22h alleles by removing one primer from each of two combinations. AccuCopy detection was used to quantify PLP products. PLP product patterns in the genotyping test allowed identifying all known Inv22. Quantitative patterns accurately represented the product patterns. The results of 164 samples detected by the genotyping test were consistent with those obtained by LD-PCR detection. Limit of detection (LOD) of the carrier mosaicism test was at least 2% of heterozygous cells with Inv22. Performing the test in two obligate mothers with negative Inv22 from two sporadic pedigrees mosaic rates of blood and hair root of the mother from pedigree 1 were 8.3% and >20%, respectively and negative results were obtained in pedigree 2. AccuCopy quantification combined with PLP (AQ-PLP) method was confirmed to be rapid and reliable for genotyping Inv22 and highly sensitive to carrier mosaicism detection. Copyright © 2016 Elsevier B.V. All rights reserved.

Development and validation of a quantitative PCR for rapid and specific detection of California sea lion adenovirus 1 and prevalence in wild and managed populations.

PubMed

Cortés-Hinojosa, Galaxia; Gulland, Frances M D; Goldstein, Tracey; Venn-Watson, Stephanie; Rivera, Rebecca; Archer, Linda L; Waltzek, Thomas B; Gray, Gregory C; Wellehan, James F X

2017-03-01

California sea lion adenovirus 1 (CSLAdV-1) has been associated with hepatitis and enteritis in several wild and captive populations of diverse pinniped species. Currently available tests have been limited to pan-adenoviral polymerase chain reaction (PCR) followed by sequencing. We present the development of a quantitative probe-hybridization PCR (qPCR) assay for rapid, sensitive, and specific detection of this virus in California sea lions ( Zalophus californianus) and other pinnipeds. This assay did not amplify other mammalian adenoviruses and is able to detect consistently down to 10 viral copies per well. Compared with the gold standard conventional pan-adenovirus PCR/sequencing assay, diagnostic sensitivity and specificity of 100% and 88.2% were found, respectively. The lower diagnostic specificity of this qPCR assay may be the result of the lower limit of detection of this assay compared with the gold standard rather than the result of detection of true false-positives.

Detection and dissemination of Toxoplasma gondii in experimentally infected calves, a single test does not tell the whole story.

PubMed

Burrells, Alison; Taroda, Alessandra; Opsteegh, Marieke; Schares, Gereon; Benavides, Julio; Dam-Deisz, Cecile; Bartley, Paul M; Chianini, Francesca; Villena, Isabella; van der Giessen, Joke; Innes, Elisabeth A; Katzer, Frank

2018-01-18

Although the detection of Toxoplasma gondii in bovine tissues is rare, beef might be an important source of human infection. The use of molecular techniques, such as magnetic capture qPCR (MC-qPCR), in combination with the gold standard method for isolating the parasite (mouse bioassay), may increase the sensitivity of T. gondii detection in infected cattle. The risk of transmission of the parasite to humans from undercooked/raw beef is not fully known and further knowledge about the predilection sites of T. gondii within cattle is needed. In the current study, six Holstein Friesian calves (Bos taurus) were experimentally infected with 10 6  T. gondii oocysts of the M4 strain and, following euthanasia (42 dpi), pooled tissues were tested for presence of the parasite by mouse bioassay and MC-qPCR. Toxoplasma gondii was detected by both MC-qPCR and mouse bioassay from distinct pools (100 g) of tissues comprising: liver, tongue, heart, diaphragm, semitendinosus (hindlimb), longissimus dorsi muscle (sirloin) and psoas major muscle (fillet). When a selection of individual tissues which had been used for mouse bioassay were examined by MC-qPCR, parasite DNA could only be detected from two animals, despite all calves showing seroconversion after infection. It is apparent that one individual test will not provide an answer as to whether a calf harbours T. gondii tissue cysts. Although the calves received a known number of infectious oocysts and highly sensitive methods for the detection of the parasite within bovine tissues were applied (mouse bioassay and MC-qPCR), the results confirm previous studies which report low presence of viable T. gondii in cattle and no clear predilection site within bovine tissues.

Detection of Francisella tularensis within infected mouse tissues by using a hand-held PCR thermocycler.

PubMed

Emanuel, Peter A; Bell, Ryan; Dang, Jessica L; McClanahan, Rebecca; David, John C; Burgess, Robert J; Thompson, Joseph; Collins, Lisa; Hadfield, Ted

2003-02-01

The diagnosis of human cases of tularemia often relies upon the demonstration of an antibody response to Francisella tularensis or the direct culturing of the bacteria from the patient. Antibody response is not detectable until 2 weeks or more after infection, and culturing requires special media and suspicion of tularemia. In addition, handling live Francisella poses a risk to laboratory personnel due to the highly infectious nature of this pathogen. In an effort to develop a rapid diagnostic assay for tularemia, we investigated the use of TaqMan 5' hydrolysis fluorogenic PCR to detect the organism in tissues of infected mice. Mice were infected to produce respiratory tularemia. The fopA and tul4 genes of F. tularensis were amplified from infected spleen, lung, liver, and kidney tissues sampled over a 5-day period. The samples were analyzed using the laboratory-based Applied Biosystems International 7900 and the Smiths Detection-Edgewood BioSeeq, a hand-held portable fluorescence thermocycler designed for use in the field. A comparison of culturing and PCR for detection of bacteria in infected tissues shows that culturing was more sensitive than PCR. However, the results for culture take 72 h, whereas PCR results were available within 4 h. PCR was able to detect infection in all the tissues tested. Lung tissue showed the earliest response at 2 days when tested with the ABI 7900 and in 3 days when tested with the BioSeeq. The results were in agreement between the ABI 7900 and the BioSeeq when presented with the same sample. Template preparation may account for the loss of sensitivity compared to culturing techniques. The hand-held BioSeeq thermocycler shows promise as an expedient means of forward diagnosis of infection in the field.

Detection of Francisella tularensis within Infected Mouse Tissues by Using a Hand-Held PCR Thermocycler

PubMed Central

Emanuel, Peter A.; Bell, Ryan; Dang, Jessica L.; McClanahan, Rebecca; David, John C.; Burgess, Robert J.; Thompson, Joseph; Collins, Lisa; Hadfield, Ted

2003-01-01

The diagnosis of human cases of tularemia often relies upon the demonstration of an antibody response to Francisella tularensis or the direct culturing of the bacteria from the patient. Antibody response is not detectable until 2 weeks or more after infection, and culturing requires special media and suspicion of tularemia. In addition, handling live Francisella poses a risk to laboratory personnel due to the highly infectious nature of this pathogen. In an effort to develop a rapid diagnostic assay for tularemia, we investigated the use of TaqMan 5′ hydrolysis fluorogenic PCR to detect the organism in tissues of infected mice. Mice were infected to produce respiratory tularemia. The fopA and tul4 genes of F. tularensis were amplified from infected spleen, lung, liver, and kidney tissues sampled over a 5-day period. The samples were analyzed using the laboratory-based Applied Biosystems International 7900 and the Smiths Detection-Edgewood BioSeeq, a hand-held portable fluorescence thermocycler designed for use in the field. A comparison of culturing and PCR for detection of bacteria in infected tissues shows that culturing was more sensitive than PCR. However, the results for culture take 72 h, whereas PCR results were available within 4 h. PCR was able to detect infection in all the tissues tested. Lung tissue showed the earliest response at 2 days when tested with the ABI 7900 and in 3 days when tested with the BioSeeq. The results were in agreement between the ABI 7900 and the BioSeeq when presented with the same sample. Template preparation may account for the loss of sensitivity compared to culturing techniques. The hand-held BioSeeq thermocycler shows promise as an expedient means of forward diagnosis of infection in the field. PMID:12574268

[A Duplex PCR Method for Detection of Babesia caballi and Theileria equi].

PubMed

Zhang, Yang; Zhang, Yu-ting; Wang, Zhen-bao; Bolati; Li, Hai; Bayinchahan

2015-04-01

To develop a duplex PCR assay for detection of Babesia caballi and Theileria equi. Two pairs of primers were designed according to the BC48 gene of B. caballi and 18 s rRNA gene of T. equi, and a duplex PCR assay was developed by the optimization of reaction conditions. The specificity, sensitivity and reliability of the method were tested. The horse blood samples of suspected cases were collected from Yili region, and detected by the duplex PCR, microspopy, conventional PCR, and fluorescence quantitative PCR, and the results were compared. Using the duplex PCR assay, the specific fragments of 155 bp and 280 bp were amplified from DNA samples of B. caballi and T. equi, respectively. No specific fragment was amplified from DNA samples of B. bigemina, Theilerdia annulata, Theilerdia sergenti, Toxoplasma gondii, Neospora caninum, and Trypanosoma evansi. The limit of detection was 4.85 x 10(5) copies/L for B. caballi DNA and 4.85 x 10(4) copies/µl for T. equi DNA, respectively. Among the 24 blood samples, 11 were found B. caballi-positive by the duplex PCR assay, and 18 were T. equi-positive. The coincidence rate of microscopy, conventional PCR, and fluorescence quantitative PCR with duplex PCR was 91.7% (22/24), 95.8% (23/24), and 95.8% (23/24), respectively. A duplex PCR assay for simultaneous detection of B. caballi and T. equi is established.

An alternative nested-PCR assay for the detection of Toxoplasma gondii strains based on GRA7 gene sequences.

PubMed

Costa, Maria Eduarda S M; Oliveira, Claudio Bruno S; Andrade, Joelma Maria de A; Medeiros, Thatiany A; Neto, Valter F Andrade; Lanza, Daniel C F

2016-07-01

Toxoplasma gondii is a widespread parasite able to infect virtually any nucleated cells of warm-blooded hosts. In some cases, T. gondii detection using already developed PCR primers can be inefficient in routine laboratory tests, especially to detect atypical strains. Here we report a new nested-PCR protocol able to detect virtually all T. gondii isolates. Analyzing 685 sequences available in GenBank, we determine that GRA7 is one of the most conserved genes of T. gondii genome. Based on an alignment of 85 GRA7 sequences new primer sets that anneal in the highly conserved regions of this gene were designed. The new GRA7 nested-PCR assay providing sensitivity and specificity equal to or greater than the gold standard PCR assays for T. gondii detection, that amplify the B1 sequence or the repetitive 529bp element. Copyright © 2016 Elsevier B.V. All rights reserved.

Detection and quantification of beef and pork materials in meat products by duplex droplet digital PCR.

PubMed

Cai, Yicun; He, Yuping; Lv, Rong; Chen, Hongchao; Wang, Qiang; Pan, Liangwen

2017-01-01

Meat products often consist of meat from multiple animal species, and inaccurate food product adulteration and mislabeling can negatively affect consumers. Therefore, a cost-effective and reliable method for identification and quantification of animal species in meat products is required. In this study, we developed a duplex droplet digital PCR (dddPCR) detection and quantification system to simultaneously identify and quantify the source of meat in samples containing a mixture of beef (Bos taurus) and pork (Sus scrofa) in a single digital PCR reaction tube. Mixed meat samples of known composition were used to test the accuracy and applicability of this method. The limit of detection (LOD) and the limit of quantification (LOQ) of this detection and quantification system were also identified. We conclude that our dddPCR detection and quantification system is suitable for quality control and routine analyses of meat products.

A sensitive duplex nanoparticle-assisted PCR assay for identifying porcine epidemic diarrhea virus and porcine transmissible gastroenteritis virus from clinical specimens.

PubMed

Zhu, Yu; Liang, Lin; Luo, Yakun; Wang, Guihua; Wang, Chunren; Cui, Yudong; Ai, Xia; Cui, Shangjin

2017-02-01

In this study, a novel duplex nanoparticle-assisted polymerase chain reaction (nanoPCR) assay was developed to detect porcine epidemic diarrhea virus (PEDV) and porcine transmissible gastroenteritis virus (TGEV). Two pairs of primers were designed based on the conserved region within the N gene of PEDV and TGEV. In a screening of 114 clinical samples from four provinces in China for PEDV and TGEV, 48.2 and 3.5 % of the samples, respectively, tested positive. Under optimized conditions, the duplex nanoPCR assay had a detection limit of 7.6 × 10 1 and 8.5 × 10 1 copies μL -1 for PEDV and TGEV, respectively. The sensitivity of the duplex nanoPCR assay was ten times higher than that of a conventional PCR assay. Moreover, no fragments were amplified when the duplex nanoPCR assay was used to test samples containing other porcine viruses. Our results indicate that the duplex nanoPCR assay described here is useful for the rapid detection of PEDV and TGEV and can be applied in clinical diagnosis.

Streamlined, PCR-based testing for pfhrp2- and pfhrp3-negative Plasmodium falciparum.

PubMed

Parr, Jonathan B; Anderson, Olivia; Juliano, Jonathan J; Meshnick, Steven R

2018-04-02

Rapid diagnostic tests (RDTs) that detect histidine-rich protein 2 (PfHRP2) are used throughout Africa for the diagnosis of Plasmodium falciparum malaria. However, recent reports indicate that parasites lacking the pfhrp2 and/or histidine-rich protein 3 (pfhrp3) genes, which produce antigens detected by these RDTs, are common in select regions of South America, Asia, and Africa. Proving the absence of a gene is challenging, and multiple PCR assays targeting these genes have been described. A detailed characterization and comparison of published assays is needed to facilitate robust and streamlined testing approaches. Among six pfhrp2 and pfhrp3 PCR assays tested, the lower limit of detection ranged from 0.01 pg/µL to 0.1 ng/µL of P. falciparum 3D7 strain DNA, or approximately 0.4-4000 parasite genomes/µL. By lowering the elongation temperature to 60 °C, a tenfold improvement in the limit of detection and/or darker bands for all exon 1 targets and for the first-round reaction of a single exon 2 target was achieved. Additionally, assays targeting exon 1 of either gene yielded spurious amplification of the paralogous gene. Using these data, an optimized testing algorithm for the detection of pfhrp2- and pfhrp3-negative P. falciparum is proposed. Surveillance of pfhrp2- and pfhrp3-negative P. falciparum requires careful laboratory workflows. PCR-based testing methods coupled with microscopy and/or antigen testing serve as useful tools to support policy development. Standardized approaches to the detection of pfhrp2- and pfhrp3-negative P. falciparum should inform efforts to define the impact of these parasites.

Validation of a Real Time PCR for Classical Swine Fever Diagnosis

PubMed Central

Dias, Natanael Lamas; Fonseca Júnior, Antônio Augusto; Oliveira, Anapolino Macedo; Sales, Érica Bravo; Alves, Bruna Rios Coelho; Dorella, Fernanda Alves

2014-01-01

The viral disease classical swine fever (CSF), caused by a Pestivirus, is one of the major causes of economic losses for pig farming. The aim of this work was to validate a RT-qPCR using Taqman for detection of CSF in swine tissues. The parameters for the validation followed the specifications of the Manual of Diagnostic Tests and Vaccines for Terrestrial Animals of the World Organization for Animal Health (OIE) and the guide ABNT NBR ISO/IEC 17025:2005. The analysis of the 5′NTR region of CSF virus was performed in 145 samples from 29 infected pigs and in 240 samples from 80 pigs originated in the Brazilian CSF-free zone. The tissues tested were spleen, kidney, blood, tonsils, and lymph nodes. Sequencing of the positive samples for 5′NTR region was performed to evaluate the specificity of the RT-qPCR. Tests performed for the RT-qPCR validation demonstrated that the PCR assay was efficient in detecting RNA from CSF virus in all materials from different tissues of infected animals. Furthermore, RNA from CSF virus was not detected in samples of swine originated from the Brazilian CSF-free zone. Hence, it is concluded that RT-qPCR can be used as a complementary diagnostic for CSF. PMID:24818039

Validation of a real time PCR for classical Swine Fever diagnosis.

PubMed

Dias, Natanael Lamas; Fonseca Júnior, Antônio Augusto; Oliveira, Anapolino Macedo; Sales, Erica Bravo; Alves, Bruna Rios Coelho; Dorella, Fernanda Alves; Camargos, Marcelo Fernandes

2014-01-01

The viral disease classical swine fever (CSF), caused by a Pestivirus, is one of the major causes of economic losses for pig farming. The aim of this work was to validate a RT-qPCR using Taqman for detection of CSF in swine tissues. The parameters for the validation followed the specifications of the Manual of Diagnostic Tests and Vaccines for Terrestrial Animals of the World Organization for Animal Health (OIE) and the guide ABNT NBR ISO/IEC 17025:2005. The analysis of the 5'NTR region of CSF virus was performed in 145 samples from 29 infected pigs and in 240 samples from 80 pigs originated in the Brazilian CSF-free zone. The tissues tested were spleen, kidney, blood, tonsils, and lymph nodes. Sequencing of the positive samples for 5'NTR region was performed to evaluate the specificity of the RT-qPCR. Tests performed for the RT-qPCR validation demonstrated that the PCR assay was efficient in detecting RNA from CSF virus in all materials from different tissues of infected animals. Furthermore, RNA from CSF virus was not detected in samples of swine originated from the Brazilian CSF-free zone. Hence, it is concluded that RT-qPCR can be used as a complementary diagnostic for CSF.

Standardisation of polymerase chain reaction for the detection of Salmonella typhi in typhoid fever.

PubMed Central

Chaudhry, R; Laxmi, B V; Nisar, N; Ray, K; Kumar, D

1997-01-01

To improve the diagnosis of Salmonella typhi infection, a polymerase chain reaction (PCR) assay was developed for the amplification of the dH flagellin gene of S typhi. Primers were designed from dH flagellin gene sequence which will give an amplification product of 486 base pairs. In tests to study the specificity of the assay, no amplification was seen in non-salmonella strains or salmonella strains with flagellar gene other than "d". Sensitivity tests determined that 28 pg of S typhi target DNA or 3 x 10(2) target bacteria could be detected by the PCR assay. Subsequently, the PCR technique was used for detection of S typhi in blood or clot cultures from 84 patients clinically suspected of having typhoid fever, and from 20 healthy control subjects. Twenty five of 84 samples from clinically suspected cases were positive by PCR; four of which were culture negative. No amplification was seen in samples from patients who were culture positive for organisms other than S typhi or from controls. The time taken for each sample for PCR analysis was less than 48 hours compared with three to five days for blood or clot culture. PCR appeared to be a promising diagnostic test for typhoid fever. Images PMID:9215131

Use of amplicon sequencing to improve sensitivity in PCR-based detection of microbial pathogen in environmental samples.

PubMed

Saingam, Prakit; Li, Bo; Yan, Tao

2018-06-01

DNA-based molecular detection of microbial pathogens in complex environments is still plagued by sensitivity, specificity and robustness issues. We propose to address these issues by viewing them as inadvertent consequences of requiring specific and adequate amplification (SAA) of target DNA molecules by current PCR methods. Using the invA gene of Salmonella as the model system, we investigated if next generation sequencing (NGS) can be used to directly detect target sequences in false-negative PCR reaction (PCR-NGS) in order to remove the SAA requirement from PCR. False-negative PCR and qPCR reactions were first created using serial dilutions of laboratory-prepared Salmonella genomic DNA and then analyzed directly by NGS. Target invA sequences were detected in all false-negative PCR and qPCR reactions, which lowered the method detection limits near the theoretical minimum of single gene copy detection. The capability of the PCR-NGS approach in correcting false negativity was further tested and confirmed under more environmentally relevant conditions using Salmonella-spiked stream water and sediment samples. Finally, the PCR-NGS approach was applied to ten urban stream water samples and detected invA sequences in eight samples that would be otherwise deemed Salmonella negative. Analysis of the non-target sequences in the false-negative reactions helped to identify primer dime-like short sequences as the main cause of the false negativity. Together, the results demonstrated that the PCR-NGS approach can significantly improve method sensitivity, correct false-negative detections, and enable sequence-based analysis for failure diagnostics in complex environmental samples. Copyright © 2018 Elsevier B.V. All rights reserved.

Comparative evaluation of diagnostic tests for the detection of Mycobacterium avium subsp. paratuberculosis in the tissues of sheep affected with distinct pathology of paratuberculosis.

PubMed

Sonawane, Ganesh G; Tripathi, Bhupendra N

2016-12-01

Paratuberculosis or Johne's disease is a chronic infectious granulomatous enteritis, mainly of cattle, sheep, goats, and other domestic and wild animals caused by Mycobacterium avium subsp. paratuberculosis (MAP). Currently, MAP has been recognized as an important animal pathogen with significant zoonotic and public health concerns. The early detection of infected animals using suitable diagnostic methods helps in developing control and preventive strategies for the herd. Therefore, the present study was aimed to determine the comparative efficacy of certain diagnostic methods used in the identification and confirmation of MAP in the ovine tissues with distinct pathology of paratuberculosis. The ileum and mesenteric lymph node (MLN) tissues were collected from 38 sheep infected with paratuberculosis from organized farms of Rajasthan. These animals were further classified as paucibacillary (PB; n=15) or multibacillary (MB; n=23) on the basis of histopathological findings and mycobacterial loads. The ileum and MLN tissues of these animals were subjected to IS900 and 251 gene polymerase chain reaction (PCR) and bacterial culture. The tissue sections from MB, PB, and uninfected control sheep groups were stained using indirect immunoperoxidase technique (IPT) and Ziehl-Neelsen (ZN) method. On bacterial culture examination of the ileum and MLN tissues using Herrold's egg yolk medium, MAP was isolated in 14 (60.9%) MB and 5 (33.3%) PB sheep. Of 38 sheep, IS900 PCR detected 21 (55.2%) positive for MAP, of which 19 (82.6%) were MB and 2 (13.3%) were PB sheep. Similarly, 251 gene PCR detected 25 (65.7%) sheep positive for MAP infection, of which 21 (91.3%) were MB and 4 (27%) were PB sheep. Thus, 251 gene PCR was found superior to IS900 PCR in the detection of MAP from the tissues. In PB sheep, IPT and ZN tests were positive in 87.5% and 50% of ileum sections and 70% and 37.5% in MLN sections, respectively. In MB sheep, IPT and ZN tests detected all animals as positive for MAP organisms or antigen and had equal sensitivity in the detection of MAP. The overall sensitivity of IPT was found superior (95%) to ZN staining (80%) in the demonstration of acid-fast bacteria or its antigen in the tissues. The sensitivity of all the tests in the detection of MAP was lower in PB sheep than in MB sheep. Bacterial culture detected MAP in only 50% of sheep and was found less sensitive than other tests used in the present study. Comparing the overall sensitivity of both the PCR assays, 251 gene PCR was found superior to IS900 gene PCR. The sensitivity of IPT was found superior (95%) to the ZN staining (80%) in the demonstration of acid-fast bacteria in the tissues. In the present study, IPT was found superior in the detection of MAP in PB and MB form of ovine paratuberculosis. This test can be used in the confirmation of post mortem diagnosis and research of paratuberculosis along with histopathology. However, 251 gene PCR assay was found easier to perform than IPT and could be used as paratuberculosis screening test in endemic sheep farms for blood and fecal samples. Copyright © 2016.

Development of a Real-Time PCR for a Sensitive One-Step Coprodiagnosis Allowing both the Identification of Carnivore Feces and the Detection of Toxocara spp. and Echinococcus multilocularis

PubMed Central

Umhang, Gérald; Poulle, Marie-Lazarine; Millon, Laurence

2016-01-01

Studying the environmental occurrence of parasites of concern for humans and animals based on coprosamples is an expanding field of work in epidemiology and the ecology of health. Detecting and quantifying Toxocara spp. and Echinococcus multilocularis, two predominant zoonotic helminths circulating in European carnivores, in feces may help to better target measures for prevention. A rapid, sensitive, and one-step quantitative PCR (qPCR) allowing detection of E. multilocularis and Toxocara spp. was developed in the present study, combined with a host fecal test based on the identification of three carnivores (red fox, dog, and cat) involved in the life cycles of these parasites. A total of 68 coprosamples were collected from identified specimens from Vulpes vulpes, Canis lupus familiaris, Canis lupus, Felis silvestris catus, Meles meles, Martes foina, and Martes martes. With DNA coprosamples, real-time PCR was performed in duplex with a qPCR inhibitor control specifically designed for this study. All the coprosample host identifications were confirmed by qPCR combined with sequencing, and parasites were detected and confirmed (E. multilocularis in red foxes and Toxocara cati in cats; 16% of samples presented inhibition). By combining parasite detection and quantification, the host fecal test, and a new qPCR inhibitor control, we created a technique with a high sensitivity that may considerably improve environmental studies of pathogens. PMID:26969697

Development of a Real-Time PCR for a Sensitive One-Step Coprodiagnosis Allowing both the Identification of Carnivore Feces and the Detection of Toxocara spp. and Echinococcus multilocularis.

PubMed

Knapp, Jenny; Umhang, Gérald; Poulle, Marie-Lazarine; Millon, Laurence

2016-05-15

Studying the environmental occurrence of parasites of concern for humans and animals based on coprosamples is an expanding field of work in epidemiology and the ecology of health. Detecting and quantifying Toxocara spp. and Echinococcus multilocularis, two predominant zoonotic helminths circulating in European carnivores, in feces may help to better target measures for prevention. A rapid, sensitive, and one-step quantitative PCR (qPCR) allowing detection of E. multilocularis and Toxocara spp. was developed in the present study, combined with a host fecal test based on the identification of three carnivores (red fox, dog, and cat) involved in the life cycles of these parasites. A total of 68 coprosamples were collected from identified specimens from Vulpes vulpes, Canis lupus familiaris, Canis lupus, Felis silvestris catus, Meles meles, Martes foina, and Martes martes With DNA coprosamples, real-time PCR was performed in duplex with a qPCR inhibitor control specifically designed for this study. All the coprosample host identifications were confirmed by qPCR combined with sequencing, and parasites were detected and confirmed (E. multilocularis in red foxes and Toxocara cati in cats; 16% of samples presented inhibition). By combining parasite detection and quantification, the host fecal test, and a new qPCR inhibitor control, we created a technique with a high sensitivity that may considerably improve environmental studies of pathogens. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

The evaluation of three diagnostic tests for the detection of equine influenza nucleoprotein in nasal swabs.

PubMed

Galvin, Pamela; Gildea, Sarah; Nelly, Maura; Quinlivan, Michelle; Arkins, Sean; Walsh, Cathal; Cullinane, Ann

2014-05-01

Equine influenza (EI) is a highly contagious respiratory disease of horses. The aim of this study was to evaluate two rapid antigen detection kits (Directigen or DFA, and Espline) and a commercial ELISA for the detection of EI nucleoprotein in nasal swabs. Nasal swab samples from naturally and experimentally infected horses were used to compare the sensitivity and specificity of these assays to virus isolation (VI) and real-time RT-PCR. If real-time RT-PCR was considered as the gold standard, the sensitivity of the other tests in field samples was 68% (DFA), 35% (ELISA), 29% (Espline), and 9% (VI). These tests had 100% specificity when compared to real-time RT-PCR. A receiver operating characteristic (ROC) curve indicated that decreasing the cutoff of the ELISA would increase sensitivity with some loss of specificity. In samples from experimentally infected horses, the sensitivity of the tests compared with real-time RT-PCR was 69% (VI), 27% (DFA), 6% (Espline), and 2% (ELISA). The specificity was 100% for Espline and ELISA and 95% for VI and DFA. This study illustrated that DFA is the most sensitive antigen detection test evaluated for the diagnosis of EI and that it can detect virus in some subclinical infected and vaccinated horses. The results suggest that DFA is a useful adjunct to laboratory tests and may be effective as a screening test in a quarantine station or similar facility where horses are monitored daily. © 2014 The Authors. Influenza and Other Respiratory Viruses Published by John Wiley & Sons Ltd.

Droplet digital PCR-based EGFR mutation detection with an internal quality control index to determine the quality of DNA.

PubMed

Kim, Sung-Su; Choi, Hyun-Jeung; Kim, Jin Ju; Kim, M Sun; Lee, In-Seon; Byun, Bohyun; Jia, Lina; Oh, Myung Ryurl; Moon, Youngho; Park, Sarah; Choi, Joon-Seok; Chae, Seoung Wan; Nam, Byung-Ho; Kim, Jin-Soo; Kim, Jihun; Min, Byung Soh; Lee, Jae Seok; Won, Jae-Kyung; Cho, Soo Youn; Choi, Yoon-La; Shin, Young Kee

2018-01-11

In clinical translational research and molecular in vitro diagnostics, a major challenge in the detection of genetic mutations is overcoming artefactual results caused by the low-quality of formalin-fixed paraffin-embedded tissue (FFPET)-derived DNA (FFPET-DNA). Here, we propose the use of an 'internal quality control (iQC) index' as a criterion for judging the minimum quality of DNA for PCR-based analyses. In a pre-clinical study comparing the results from droplet digital PCR-based EGFR mutation test (ddEGFR test) and qPCR-based EGFR mutation test (cobas EGFR test), iQC index ≥ 0.5 (iQC copies ≥ 500, using 3.3 ng of FFPET-DNA [1,000 genome equivalents]) was established, indicating that more than half of the input DNA was amplifiable. Using this criterion, we conducted a retrospective comparative clinical study of the ddEGFR and cobas EGFR tests for the detection of EGFR mutations in non-small cell lung cancer (NSCLC) FFPET-DNA samples. Compared with the cobas EGFR test, the ddEGFR test exhibited superior analytical performance and equivalent or higher clinical performance. Furthermore, iQC index is a reliable indicator of the quality of FFPET-DNA and could be used to prevent incorrect diagnoses arising from low-quality samples.

TaqMan 5′-Nuclease Human Immunodeficiency Virus Type 1 PCR Assay with Phage-Packaged Competitive Internal Control for High-Throughput Blood Donor Screening

PubMed Central

Drosten, C.; Seifried, E.; Roth, W. K.

2001-01-01

Screening of blood donors for human immunodeficiency virus type 1 (HIV-1) infection by PCR permits the earlier diagnosis of HIV-1 infection compared with that by serologic assays. We have established a high-throughput reverse transcription (RT)-PCR assay based on 5′-nuclease PCR. By in-tube detection of HIV-1 RNA with a fluorogenic probe, the 5′-nuclease PCR technology (TaqMan PCR) eliminates the risk of carryover contamination, a major problem in PCR testing. We outline the development and evaluation of the PCR assay from a technical point of view. A one-step RT-PCR that targets the gag genes of all known HIV-1 group M isolates was developed. An internal control RNA detectable with a heterologous 5′-nuclease probe was derived from the viral target cDNA and was packaged into MS2 coliphages (Armored RNA). Because the RNA was protected against digestion with RNase, it could be spiked into patient plasma to control the complete sample preparation and amplification process. The assay detected 831 HIV-1 type B genome equivalents per ml of native plasma (95% confidence interval [CI], 759 to 936 HIV-1 B genome equivalents per ml) with a ≥95% probability of a positive result, as determined by probit regression analysis. A detection limit of 1,195 genome equivalents per ml of (individual) donor plasma (95% CI, 1,014 to 1,470 genome equivalents per ml of plasma pooled from individuals) was achieved when 96 samples were pooled and enriched by centrifugation. Up to 4,000 plasma samples per PCR run were tested in a 3-month trial period. Although data from the present pilot feasibility study will have to be complemented by a large clinical validation study, the assay is a promising approach to the high-throughput screening of blood donors and is the first noncommercial test for high-throughput screening for HIV-1. PMID:11724836

Utilization of a DNA enzyme immunoassay for the detection of proviral DNA of human immunodeficiency virus type 1 by polymerase chain reaction.

PubMed

Zella, D; Cavicchini, A; Cattaneo, E; Cimarelli, A; Bertazzoni, U

1995-02-01

The detection of proviral DNA by Polymerase Chain Reaction (PCR) is regarded as an important tool in the diagnosis of HIV-1 infection, specially among adults at risk of AIDS and children born to seropositive mothers. However, application of PCR in routine testing is hampered by the need to use radioactive probes. In this study, a non-radioactive test based on a microtiter plate (DNA Enzyme ImmunoAssay, DEIA) was used for the detection of proviral sequences of HIV-1 in peripheral blood cells of different patients. The results of the PCR-DEIA assay were compared to those obtained by liquid hybridization (PCR-LH), virus isolation (VI) and Western blot (WB). The study population included 92 patients belonging to three different groups: seropositive subjects with a well-defined clinical status and WB profile; adults at risk of infection with negative or indeterminate WB; children born to seropositive mothers with still unestablished HIV-1 infection. In the seropositive subjects, both PCR-LH and PCR-DEIA confirmed infection and gave the same results as WB. In adults at risk of infection, PCR with both methods anticipated the seroconversion in one patient with indeterminate WB and confirmed the absence of infection among seronegative and other indeterminate patients. In children born to seropositive mothers, both PCR systems as well as VI permitted an early diagnosis of infection, as confirmed by the clinical follow-up. This study has shown that in subjects at risk of AIDS and in children born to seropositive mothers, the non-isotopic DEIA method presents the same sensitivity and specificity for the detection of HIV-1 infection as the radioactive procedure. The DEIA method appears to be particularly useful for the detection of PCR products in routine diagnostic analyses.

Development and application of absolute quantitative detection by duplex chamber-based digital PCR of genetically modified maize events without pretreatment steps.

PubMed

Zhu, Pengyu; Fu, Wei; Wang, Chenguang; Du, Zhixin; Huang, Kunlun; Zhu, Shuifang; Xu, Wentao

2016-04-15

The possibility of the absolute quantitation of GMO events by digital PCR was recently reported. However, most absolute quantitation methods based on the digital PCR required pretreatment steps. Meanwhile, singleplex detection could not meet the demand of the absolute quantitation of GMO events that is based on the ratio of foreign fragments and reference genes. Thus, to promote the absolute quantitative detection of different GMO events by digital PCR, we developed a quantitative detection method based on duplex digital PCR without pretreatment. Moreover, we tested 7 GMO events in our study to evaluate the fitness of our method. The optimized combination of foreign and reference primers, limit of quantitation (LOQ), limit of detection (LOD) and specificity were validated. The results showed that the LOQ of our method for different GMO events was 0.5%, while the LOD is 0.1%. Additionally, we found that duplex digital PCR could achieve the detection results with lower RSD compared with singleplex digital PCR. In summary, the duplex digital PCR detection system is a simple and stable way to achieve the absolute quantitation of different GMO events. Moreover, the LOQ and LOD indicated that this method is suitable for the daily detection and quantitation of GMO events. Copyright © 2016 Elsevier B.V. All rights reserved.

[Evaluation of the new ImmunoCard STAT!® CGE test for the diagnosis of Amebiasis].

PubMed

Formenti, F; Perandin, F; Bonafini, S; Degani, M; Bisoffi, Z

2015-08-01

For many years, microscopic examination of stool samples has been considered to be the "gold standard" for diagnosis of intestinal parasites although the Polymerase Chain Reaction (PCR) analysis is increasingly utilized due to its high accuracy. Recently, PCR has been approved by the World Health Organization as the current method of choice for the diagnosis of Entamoeba histolytica infection. In this study we evaluated a novel immunochromatographic antigen detection rapid test, ImmunoCardSTAT CGE (Meridian Bioscence, Milan, Italy), which has been proposed for the diagnosis of infections caused by Cryptosporidium parvum-Giardia intestinalis-Entamoeba histolytica. There is another rapid test with a similar name, the ImmunoCard STAT! Crypto/Giardia, but it is just for Cryptosporidium and Giardia. We aimed to compare E. histolytica results obtained from the rapid test with those of a rt-PCR for the detection of E. histolytica / E. dispar DNA. The new ImmunoCard rapid antigen detection test exhibited 88% sensitivity and 92% specificity (if assessed on rt-PCR negative samples) but showed a high proportion of cross-reaction between the pathogenic E. histolytica and the non pathogenic E. dispar.

Development and evaluation of tailored specific real-time RT-PCR assays for detection of foot-and-mouth disease virus serotypes circulating in East Africa.

PubMed

Bachanek-Bankowska, Katarzyna; Mero, Herieth R; Wadsworth, Jemma; Mioulet, Valerie; Sallu, Raphael; Belsham, Graham J; Kasanga, Christopher J; Knowles, Nick J; King, Donald P

2016-11-01

Rapid, reliable and accurate diagnostic methods provide essential support to programmes that monitor and control foot-and-mouth disease (FMD). While pan-specific molecular tests for FMD virus (FMDV) detection are well established and widely used in endemic and FMD-free countries, current serotyping methods mainly rely either on antigen detection ELISAs or nucleotide sequencing approaches. This report describes the development of a panel of serotype-specific real-time RT-PCR assays (rRT-PCR) tailored to detect FMDV lineages currently circulating in East Africa. These assays target sequences within the VP1-coding region that share high intra-lineage identity, but do not cross-react with FMD viruses from other serotypes that circulate in the region. These serotype-specific assays operate with the same thermal profile as the pan-diagnostic tests making it possible to run them in parallel to produce C T values comparable to the pan-diagnostic test detecting the 3D-coding region. These assays were evaluated alongside the established pan-specific molecular test using field samples and virus isolates collected from Tanzania, Kenya and Ethiopia that had been previously characterised by nucleotide sequencing. Samples (n=71) representing serotype A (topotype AFRICA, lineage G-I), serotype O (topotypes EA-2 and EA-4), serotype SAT 1 (topotype I (NWZ)) and serotype SAT2 (topotype IV) were correctly identified with these rRT-PCR assays. Furthermore, FMDV RNA from samples that did not contain infectious virus could still be serotyped using these assays. These serotype-specific real-time RT-PCR assays can detect and characterise FMDVs currently circulating in East Africa and hence improve disease control in this region. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Evaluation of a manual DNA extraction protocol and an isothermal amplification assay for detecting HIV-1 DNA from dried blood spots for use in resource-limited settings.

PubMed

Jordan, Jeanne A; Ibe, Christine O; Moore, Miranda S; Host, Christel; Simon, Gary L

2012-05-01

In resource-limited settings (RLS) dried blood spots (DBS) are collected on infants and transported through provincial laboratories to a central facility where HIV-1 DNA PCR testing is performed using specialized equipment. Implementing a simpler approach not requiring such equipment or skilled personnel could allow the more numerous provincial laboratories to offer testing, improving turn-around-time to identify and treat infected infants sooner. Assess performances of a manual DNA extraction method and helicase-dependent amplification (HDA) assay for detecting HIV-1 DNA from DBS. 60 HIV-1 infected adults were enrolled, blood samples taken and DBS made. DBS extracts were assessed for DNA concentration and beta globin amplification using PCR and melt-curve analysis. These same extracts were then tested for HIV-1 DNA using HDA and compared to results generated by PCR and pyrosequencing. Finally, HDA limit of detection (LOD) studies were performed using DBS extracts prepared with known numbers of 8E5 cells. The manual extraction protocol consistently yielded high concentrations of amplifiable DNA from DBS. LOD assessment demonstrated HDA detected ∼470 copies/ml of HIV-1 DNA extracts in 4/4 replicates. No statistical difference was found using the McNemar's test when comparing HDA to PCR for detecting HIV-1 DNA from DBS. Using just a magnet, heat block and pipettes, the manual extraction protocol and HDA assay detected HIV-1 DNA from DBS at levels that would be useful for early infant diagnosis. Next steps will include assessing HDA for non-B HIV-1 subtypes recognition and comparison to Roche HIV-1 DNA v1.5 PCR assay. Copyright © 2012 Elsevier B.V. All rights reserved.

A serotype-specific polymerase chain reaction for identification of Pasteurella multocida serotype 1

USGS Publications Warehouse

Rocke, Tonie E.; Smith, Susan R.; Miyamoto, Amy; Shadduck, Daniel J.

2002-01-01

A serotype-specific polymerase chain reaction (PCR) assay was developed for detection and identification of Pasteurella multocida serotype 1, the causative agent of avian cholera in wild waterfowl. Arbitrarily primed PCR was used to detect DNA fragments that distinguish serotype 1 from the other 15 serotypes of P. multocida (with the exception of serotype 14). Oligonucleotide primers were constructed from these sequences, and a PCR assay was optimized and evaluated. PCR reactions consistently resulted in amplification products with reference strains 1 and 14 and all other serotype 1 strains tested, with cell numbers as low as 2.3 cells/ml. No amplification products were produced with other P. multocida serotypes or any other bacterial species tested. To compare the sensitivity and further test the specificity of this PCR assay with traditional culturing and serotyping techniques, tissue samples from 84 Pekin ducks inoculated with field strains of P. multocida and 54 wild lesser snow geese collected during an avian cholera outbreak were provided by other investigators working on avian cholera. PCR was as sensitive (58/64) as routine isolation (52/64) in detecting and identifying P. multocida serotype 1 from the livers of inoculated Pekins that became sick or died from avian cholera. No product was amplified from tissues of 20 other Pekin ducks that received serotypes other than type 1 (serotype 3, 12 × 3, or 10) or 12 control birds. Of the 54 snow geese necropsied and tested for P. multocida, our PCR detected and identified the bacteria from 44 compared with 45 by direct isolation. The serotype-specific PCR we developed was much faster and less labor intensive than traditional culturing and serotyping procedures and could result in diagnosis of serotype 1 pasteurellosis within 24 hr of specimen submission.

Molecular diagnostics for human leptospirosis.

PubMed

Waggoner, Jesse J; Pinsky, Benjamin A

2016-10-01

The definitive diagnosis of leptospirosis, which results from infection with spirochetes of the genus Leptospira, currently relies on the use of culture, serological testing (microscopic agglutination testing), and molecular detection. The purpose of this review is to describe new molecular diagnostics for Leptospira and discuss advancements in the use of available methods. Efforts have been focused on improving the clinical sensitivity of Leptospira detection using molecular methods. In this review, we describe a reoptimized pathogenic species-specific real-time PCR (targeting lipL32) that has demonstrated improved sensitivity, findings by two groups that real-time reverse-transcription PCR assays targeting the 16S rrs gene can improve detection, and two new loop-mediated amplification techniques. Quantitation of leptospiremia, detection in different specimen types, and the complementary roles played by molecular detection and microscopic agglutination testing will be discussed. Finally, a protocol for Leptospira strain subtyping using variable number tandem repeat targets and high-resolution melting will be described. Molecular diagnostics have an established role for the diagnosis of leptospirosis and provide an actionable diagnosis in the acute setting. The use of real-time reverse-transcription PCR for testing serum/plasma and cerebrospinal fluid, when available, may improve the detection of Leptospira without decreasing clinical specificity.

Accurate clinical genetic testing for autoinflammatory diseases using the next-generation sequencing platform MiSeq.

PubMed

Nakayama, Manabu; Oda, Hirotsugu; Nakagawa, Kenji; Yasumi, Takahiro; Kawai, Tomoki; Izawa, Kazushi; Nishikomori, Ryuta; Heike, Toshio; Ohara, Osamu

2017-03-01

Autoinflammatory diseases occupy one of a group of primary immunodeficiency diseases that are generally thought to be caused by mutation of genes responsible for innate immunity, rather than by acquired immunity. Mutations related to autoinflammatory diseases occur in 12 genes. For example, low-level somatic mosaic NLRP3 mutations underlie chronic infantile neurologic, cutaneous, articular syndrome (CINCA), also known as neonatal-onset multisystem inflammatory disease (NOMID). In current clinical practice, clinical genetic testing plays an important role in providing patients with quick, definite diagnoses. To increase the availability of such testing, low-cost high-throughput gene-analysis systems are required, ones that not only have the sensitivity to detect even low-level somatic mosaic mutations, but also can operate simply in a clinical setting. To this end, we developed a simple method that employs two-step tailed PCR and an NGS system, MiSeq platform, to detect mutations in all coding exons of the 12 genes responsible for autoinflammatory diseases. Using this amplicon sequencing system, we amplified a total of 234 amplicons derived from the 12 genes with multiplex PCR. This was done simultaneously and in one test tube. Each sample was distinguished by an index sequence of second PCR primers following PCR amplification. With our procedure and tips for reducing PCR amplification bias, we were able to analyze 12 genes from 25 clinical samples in one MiSeq run. Moreover, with the certified primers designed by our short program-which detects and avoids common SNPs in gene-specific PCR primers-we used this system for routine genetic testing. Our optimized procedure uses a simple protocol, which can easily be followed by virtually any office medical staff. Because of the small PCR amplification bias, we can analyze simultaneously several clinical DNA samples with low cost and can obtain sufficient read numbers to detect a low level of somatic mosaic mutations.

Comparison of Simplexa HSV 1 & 2 PCR with culture, immunofluorescence, and laboratory-developed TaqMan PCR for detection of herpes simplex virus in swab specimens.

PubMed

Gitman, Melissa R; Ferguson, David; Landry, Marie L

2013-11-01

The Simplexa HSV 1 & 2 direct PCR assay was compared with conventional cell culture, cytospin-enhanced direct fluorescent antibody (DFA), and a laboratory-developed real-time TaqMan PCR (LDT HSV PCR) using extracted nucleic acid for the detection of herpes simplex virus (HSV) in dermal, genital, mouth, ocular, and other swab samples. One hundred seventy-one swabs were tested prospectively, and 58 were positive for HSV (34 HSV-1 and 24 HSV-2). Cytospin-DFA detected 50 (86.2%), conventional cell culture 51 (87.9%), Simplexa direct 55 (94.8%), and LDT HSV PCR 57 (98.3%) of 58 true positives. Simplexa direct detected more positives than DFA and culture, but the differences were not significant (P = 0.0736 and P = 0.3711, respectively, by the McNemar test). Samples that were positive by all methods (n = 48) were strong positives (LDT cycle threshold [CT] value, 14.4 to 26.1). One strongly positive sample was falsely negative by LDT HSV PCR due to a failure of TaqMan probe binding. Three samples falsely negative by Simplexa direct had high CT values by LDT HSV PCR (LDT CT, 35.8 to 38.2). Omission of the DNA extraction step by Simplexa direct led to a drop in sensitivity compared to the sensitivity of LDT HSV PCR using extracted samples (94.8% versus 98.3%, respectively), but the difference was not significant (P = 0.6171). Simplexa HSV 1 & 2 direct PCR was the most expensive but required the least training of the assays used, had the lowest hands-on time and fastest assay time (75 min, versus 3 h by LDT HSV PCR), and provided the HSV type.

Comparison of Simplexa HSV 1 & 2 PCR with Culture, Immunofluorescence, and Laboratory-Developed TaqMan PCR for Detection of Herpes Simplex Virus in Swab Specimens

PubMed Central

Gitman, Melissa R.; Ferguson, David

2013-01-01

The Simplexa HSV 1 & 2 direct PCR assay was compared with conventional cell culture, cytospin-enhanced direct fluorescent antibody (DFA), and a laboratory-developed real-time TaqMan PCR (LDT HSV PCR) using extracted nucleic acid for the detection of herpes simplex virus (HSV) in dermal, genital, mouth, ocular, and other swab samples. One hundred seventy-one swabs were tested prospectively, and 58 were positive for HSV (34 HSV-1 and 24 HSV-2). Cytospin-DFA detected 50 (86.2%), conventional cell culture 51 (87.9%), Simplexa direct 55 (94.8%), and LDT HSV PCR 57 (98.3%) of 58 true positives. Simplexa direct detected more positives than DFA and culture, but the differences were not significant (P = 0.0736 and P = 0.3711, respectively, by the McNemar test). Samples that were positive by all methods (n = 48) were strong positives (LDT cycle threshold [CT] value, 14.4 to 26.1). One strongly positive sample was falsely negative by LDT HSV PCR due to a failure of TaqMan probe binding. Three samples falsely negative by Simplexa direct had high CT values by LDT HSV PCR (LDT CT, 35.8 to 38.2). Omission of the DNA extraction step by Simplexa direct led to a drop in sensitivity compared to the sensitivity of LDT HSV PCR using extracted samples (94.8% versus 98.3%, respectively), but the difference was not significant (P = 0.6171). Simplexa HSV 1 & 2 direct PCR was the most expensive but required the least training of the assays used, had the lowest hands-on time and fastest assay time (75 min, versus 3 h by LDT HSV PCR), and provided the HSV type. PMID:24006008

Evaluation of Line Immunoassay to Detect HTLV-1 Infection in an Endemic Area, Southwestern Japan; Comparison with Polymerase Chain Reaction and Western Blot.

PubMed

Umeki, Kazumi; Umekita, Kunihiko; Hashikura, Yuuki; Yamamoto, Ikuo; Kubo, Kazuyoshi; Nagatomo, Yasuhiro; Okayama, Akihiko

2017-02-01

Human T-lymphotropic virus type 1 (HTLV-1) has been recognized as a cause of adult T-cell leukemia/lymphoma, HTLV-1-associated myelopathy/tropical spastic paraparesis, and HTLV-1-associated uveitis. HTLV-1 infection is normally detected by screening for HTLV-1 antibodies, and positive samples are confirmed by Western blot (WB). However, WB fails to confirm some samples that were positive for HTLV-1 antibodies on screening. Line immunoassay (LIA) is commonly used in Europe and Brazil, but not in Japan. Therefore, we evaluated the performance of LIA as a method of confirming HTLV-1 antibodies using samples in Japan. LIA was compared with polymerase chain reaction (PCR) and WB using 50 negative and 70 positive samples tested by chemiluminescent enzyme immunoassay (CLEIA) in Miyazaki, Japan, an HTLV-1 endemic area. LIA (INNO-LIA HTLVI/II Score) and WB (Problot HTLV-I) were performed according to the manufacturer's instructions. Real-time PCR for HTLV-1 pX region was performed using DNA derived from white blood cells. The samples that tested negative by real-time PCR were further tested by nested PCR. All 50 CLEIA negative samples were determined to be negative by LIA and PCR. Of the 70 positive samples, 66 tested positive by both of LIA and PCR. Three samples tested negative by LIA and PCR, and the remaining sample (PCR negative) showed non-specific staining in LIA and WB. WB showed more indeterminate results than LIA. Gp21 antibody in LIA demonstrated a high ability to discriminate between positive and negative PCR results. Furthermore, the degree of gp21 antibody reaction by LIA showed correlation with HTLV-1 proviral loads (PVLs). Our results indicate that LIA performs well in confirming HTLV-1 seropositivity by showing a low incidence of indeterminate results and good agreement with PCR using samples in Japan, although the number of samples tested was small. In addition, semi-quantitative antibody titer to gp21 correlated well with HTLV-1 PVLs. Further study including larger samples is necessary to determine the positioning of LIA for HTLV-1 detection in Japan.

Diagnostic value of nested-PCR for identification of Malassezia species in dandruff

NASA Astrophysics Data System (ADS)

Jusuf, N. K.; Nasution, T. A.; Ullyana, S.

2018-03-01

Dandruff or pityriasis simplex is a condition of abnormal occurrence of formation of yellowish white scales from the scalp. Many factors play a role in the pathogenesis of dandruff, i.e.colonization of Malassezia species. Examination of Malassezia species previously done by culture as the gold standard. However, there are various difficulties in doing the culture. Identification method with anested-polymerase chain reaction (nested-PCR) is expected to provide quickly and easily detected. This study aimedto determine the diagnostic value of nested-PCR in the identification of Malassezia species in dandruff. From 21 subjects, scales from the scalp were taken and sent to the laboratory for nested-PCR identification. Statistical analysis of diagnostic test carried out to determine sensitivity, specificity, positive predictive value, and negative predictive value. The results showed nested-PCR detected 10 sample (47.6%) positive for Malassezia species consist of M. sympodialis (23.8%); M. slooffiae (9.5%); M. furfur (4.8%); M. globosa and M. furfur (4.8%); and M. restricta and M. sympodialis (4.8%). Detection of Malassezia species by nested-PCR has 100% in sensitivity whereas the specificity was 55%. Nested-PCR test has high sensitivity. Therefore nested-PCR may be considered for a faster and simpler alternative examination in identification for Malassezia species in dandruff.

Efficacy of the detection of Legionella in hot and cold water samples by culture and PCR. I. Standardization of methods.

PubMed

Wójcik-Fatla, Angelina; Stojek, Nimfa Maria; Dutkiewicz, Jacek

2012-01-01

The aim of the present study was: - to compare methods for concentration and isolation of Legionella DNA from water; - to examine the efficacy of various modifications of PCR test (PCR, semi-nested PCR, and real-time PCR) for the detection of known numbers of Legionella pneumophila in water samples artificially contaminated with the strain of this bacterium and in randomly selected samples of environmental water, in parallel with examination by culture. It was found that filtration is much more effective than centrifugation for the concentration of DNA in water samples, and that the Qiamp DNA Mini-Kit is the most efficient for isolation of Legionella DNA from water. The semi-nested PCR and real-time PCR proved to be the most sensitive methods for detection of Legionella DNA in water samples. Both PCR modifications showed a high correlation with recovery of Legionella by culture (p<0.01), while no correlation occurred between the results of one-stage PCR and culture (p>0.1).

Development of a highly sensitive one-tube nested real-time PCR for detecting Mycobacterium tuberculosis.

PubMed

Choi, Yeonim; Jeon, Bo-Young; Shim, Tae Sun; Jin, Hyunwoo; Cho, Sang-Nae; Lee, Hyeyoung

2014-12-01

Rapid, accurate detection of Mycobacterium tuberculosis is crucial in the diagnosis of tuberculosis (TB), but conventional diagnostic methods have limited sensitivity and specificity or are time consuming. A new highly sensitive nucleic acid amplification test, combined nested and real-time polymerase chain reaction (PCR) in a single tube (one-tube nested real-time PCR), was developed for detecting M. tuberculosis, which takes advantage of two PCR techniques, i.e., nested PCR and real-time PCR. One-tube nested real-time PCR was designed to have two sequential reactions with two sets of primers and dual probes for the insertion sequence (IS) 6110 sequence of M. tuberculosis in a single closed tube. The minimum limits of detection of IS6110 real-time PCR and IS6110 one-tube nested real-time PCR were 100 fg/μL and 1 fg/μL of M. tuberculosis DNA, respectively. AdvanSure TB/non-tuberculous mycobacteria (NTM) real-time PCR, IS6110 real-time PCR, and two-tube nested real-time PCR showed 100% sensitivity and 100% specificity for clinical M. tuberculosis isolates and NTM isolates. In comparison, the sensitivities of AdvanSure TB/NTM real-time PCR, single IS6110 real-time PCR, and one-tube nested real-time PCR were 91% (152/167), 94.6% (158/167), and 100% (167/167) for sputum specimens, respectively. In conclusion, IS6110 one-tube nested real-time PCR is useful for detecting M. tuberculosis due to its high sensitivity and simple manipulation. Copyright © 2014 Elsevier Inc. All rights reserved.

Evaluation of a PCR Test for Detection of Treponema pallidum in Swabs and Blood

PubMed Central

Grange, P. A.; Gressier, L.; Dion, P. L.; Farhi, D.; Benhaddou, N.; Gerhardt, P.; Morini, J. P.; Deleuze, J.; Pantoja, C.; Bianchi, A.; Lassau, F.; Avril, M. F.; Janier, M.

2012-01-01

Syphilis diagnosis is based on clinical observation, serological analysis, and dark-field microscopy (DFM) detection of Treponema pallidum subsp. pallidum, the etiological agent of syphilis, in skin ulcers. We performed a nested PCR (nPCR) assay specifically amplifying the tpp47 gene of T. pallidum from swab and blood specimens. We studied a cohort of 294 patients with suspected syphilis and 35 healthy volunteers. Eighty-seven of the 294 patients had primary syphilis, 103 had secondary syphilis, 40 had latent syphilis, and 64 were found not to have syphilis. The T. pallidum nPCR results for swab specimens were highly concordant with syphilis diagnosis, with a sensitivity of 82% and a specificity of 95%. Reasonable agreement was observed between the results obtained with the nPCR and DFM methods (kappa = 0.53). No agreement was found between the nPCR detection of T. pallidum in blood and the diagnosis of syphilis, with sensitivities of 29, 18, 14.7, and 24% and specificities of 96, 92, 93, and 97% for peripheral blood mononuclear cell (PBMC), plasma, serum, and whole-blood fractions, respectively. HIV status did not affect the frequency of T. pallidum detection in any of the specimens tested. Swab specimens from mucosal or skin lesions seemed to be more useful than blood for the efficient detection of the T. pallidum genome and, thus, for the diagnosis of syphilis. PMID:22219306

Development of a PCR protocol to detect aflatoxigenic molds in food products.

PubMed

Luque, M Isabel; Rodríguez, Alicia; Andrade, María J; Martín, Alberto; Córdoba, Juan J

2012-01-01

Aflatoxins are secondary metabolites produced mainly by Aspergillus species growing in foodstuffs. Because aflatoxins have important health effects, the detection of early contamination of foods by aflatoxigenic molds should be useful. In the present work, a reliable conventional PCR method for detecting aflatoxigenic molds of various species was developed. Fifty-six aflatoxigenic and nonaflatoxigenic strains commonly reported in foodstuffs were tested. Aflatoxin production was first confirmed by micellar electrokinetic capillary electrophoresis or/and high-pressure liquid chromatography-mass spectrometry. Based on the conserved regions of the O-methyltransferase gene (omt-1) involved in the aflatoxin biosynthetic pathway, six primer pairs were designed. With only the designed primer pair AFF1-AFR3, the expected PCR product (381 bp) was obtained in all of the tested aflatoxigenic strains of various species and genera. Amplification products were not obtained with this primer pair for any of the nonaflatoxigenic reference molds. However, an amplicon of 453 bp was obtained for all aflatoxigenic and nonaflatoxigenic mold reference strains with a PCR protocol based on the constitutive fungal β-tubulin gene, which was used as a positive fungal control. The PCR protocol based on omt-1 detected as little as 15 pg of DNA from aflatoxigenic molds and 10(2) to 10(3) CFU/g in contaminated food samples. This PCR protocol should be used as a routine technique to detect aflatoxigenic molds in foods.

Development of a nested polymerase chain reaction for amplification of a sequence of the p57 gene of Renibacterium salmoninarum that provides a highly sensitive method for detection of the bacterium in salmonid kidney

USGS Publications Warehouse

Chase, D.M.; Pascho, R.J.

1998-01-01

Nucleic acid-based assays have shown promise for diagnosing Renibacterium salmoninarum in tissues and body fluids of salmonids. DeVelopment of a nested polymerase chain reaction (PCR) method to detect a 320 bp DNA segment of the gene encoding the p57 protein of R. salmoninarum is described. Whereas a conventional PCR for a 383 bp segment of the p57 gene reliably detected 1000 R. salmoninarum cells per reaction in kidney tissue, the nested PCR detected as few as 10 R. salmoninarum per reaction in kidney tissue. Two DNA extraction methods for the nested PCR were compared and the correlation between replicate samples was generally higher in samples extracted by the QIAamp system compared with those extracted by the phenol/chloroform method. The specificity of the nested PCR was confirmed by testing DNA extracts of common bacterial fish pathogens and a panel of bacterial species reported to cause false-positive reactions in the enzyme-linked immunosorbent assay (ELISA) and the fluorescent antibody test (FAT) for R. salmoninarum. Kidney samples from 74 naturally infected chinook Salmon were examined by the nested PCR, the ELISA, and the FAT, and the detected prevalences of R. salmoninarum were 61, 47, and 43%, respectively.

New PCR diagnostic systems for the detection and quantification of porcine cytomegalovirus (PCMV).

PubMed

Morozov, Vladimir A; Morozov, Alexey V; Denner, Joachim

2016-05-01

Pigs are frequently infected with porcine cytomegalovirus (PCMV). Infected adult animals may not present with symptoms of disease, and the virus remains latent. However, the virus may be transmitted to human recipients receiving pig transplants. Recently, it was shown that pig-to-non-human-primate xenotransplantations showed 2 to 3 times lower transplant survival when the donor pig was infected with PCMV. Therefore, highly sensitive methods are required to select virus-free pigs and to examine xenotransplants. Seven previously established PCR detection systems targeting the DNA polymerase gene of PCMV were examined by comparison of thermodynamic parameters of oligonucleotides, and new diagnostic nested PCR and real-time PCR systems with improved parameters and high sensitivity were established. The detection limit of conventional PCR was estimated to be 15 copies, and that of the nested PCR was 5 copies. The sensitivity of the real-time PCR with a TaqMan probe was two copies. An equal efficiency of the newly established detection systems was shown by parallel testing of DNA from sera and blood of six pigs, identifying the same animals as PCMV infected. These new diagnostic PCR systems will improve the detection of PCMV and therefore increase the safety of porcine xenotransplants.

Detection of pseudorabies virus by duplex droplet digital PCR assay.

PubMed

Ren, Meishen; Lin, Hua; Chen, Shijie; Yang, Miao; An, Wei; Wang, Yin; Xue, Changhua; Sun, Yinjie; Yan, Yubao; Hu, Juan

2018-01-01

Aujeszky's disease, caused by pseudorabies virus (PRV), has damaged the economy of the Chinese swine industry. A large number of PRV gene-deleted vaccines have been constructed based on deletion of the glycoprotein E ( gE) gene combined with other virulence-related gene deletions, such as thymidine kinase ( TK), whereas PRV wild-type strains contain an intact gE gene. We developed a sensitive duplex droplet digital PCR (ddPCR) assay to rapidly detect PRV wild-type isolates and gE gene-deleted viral vaccines. We compared this assay with a TaqMan real-time PCR (qPCR) using the same primers and probes. Both assays exhibited good linearity and repeatability; however, ddPCR maintained linearity at extremely low concentrations, whereas qPCR did not. Based on positive results for both gE and gB, the detection limit of ddPCR was found to be 4.75 copies/µL in contrast of 76 copies/µL for qPCR, showing that ddPCR provided a 16-fold improvement in sensitivity. In addition, no nonspecific amplification was shown in specificity testing, and the PRV wild-type was distinguished from a gE-deleted strain. The ddPCR was more sensitive when analyzing clinical serum samples. Thus, ddPCR may become an appropriate detection platform for PRV.

Highly Sensitive Assay for Detection of Enterovirus in Clinical Specimens by Reverse Transcription-PCR with an Armored RNA Internal Control

PubMed Central

Beld, Marcel; Minnaar, René; Weel, Jan; Sol, Cees; Damen, Marjolein; van der Avoort, Harry; Wertheim-van Dillen, Pauline; Breda, Alex van; Boom, René

2004-01-01

The objective of the present study was the development of a diagnostic reverse transcription (RT)-PCR for the specific detection of enterovirus (EV) RNA in clinical specimens controlled by an internal control (IC) RNA. The IC RNA contains the same primer binding sites as EV RNA but has a different probe region. The IC RNA was packaged into an MS2 phage core particle (armored) and was added to the clinical sample to allow monitoring of both extraction efficiency and RT-PCR efficiency. Serial dilutions of the IC RNA were made, and the detection limit of the RT-PCR was tested in a background of EV RNA-negative cerebrospinal fluid. The sensitivity and specificity of the RT-PCR assay were tested by using all 64 known EV serotypes, several non-EV serotypes, and two Quality Control for Molecular Diagnostics (QCMD) Program EV proficiency panels from 2001 and 2002. In total, 322 clinical specimens were tested by RT-PCR, and to establish the clinical utility of the RT-PCR, a comparison of the results of viral culture and RT-PCR was done with 87 clinical specimens. The lower limit of sensitivity was reached at about 150 copies of IC RNA/ml. All 64 EV serotypes were positive, while all non-EV serotypes were negative. All culture-positive samples of the 2001 QCMD proficiency panel (according to the 50% tissue culture infective doses per milliliter) were positive by RT-PCR. Invalid results, i.e., negativity for both EV RNA and IC RNA, due to inhibition of RT-PCR were observed for 33.3% of the members of the 2002 QCMD proficiency panel and 3.1% of the clinical specimens. Inhibition of RT-PCR could be relieved by the addition of 400 ng of bovine α-casein per μl to both the RT reaction mixture and the PCR mixture. With this optimized protocol, the results for all samples of the 2002 QCMD proficiency panel and all clinical specimens except one fecal sample (0.3%) were valid. Evaluation of the clinical samples demonstrated that EV infection could be detected in 12 of 87 samples (13.8%) by RT-PCR, while viral culture was negative. Our data show that the RT-PCR with armored IC RNA offers a very reliable and rapid diagnostic tool for the detection of EV in clinical specimens and that the addition of bovine α-casein relieved inhibition of the RT-PCR for 99.7% of clinical specimens. PMID:15243060

Accuracy of polimerase chain reaction for the diagnosis of pleural tuberculosis.

PubMed

Trajman, Anete; da Silva Santos Kleiz de Oliveira, Elen Fabricia; Bastos, Mayara Lisboa; Belo Neto, Epaminondas; Silva, Edgar Manoel; da Silva Lourenço, Maria Cristina; Kritski, Afrânio; Oliveira, Martha Maria

2014-06-01

Polymerase chain reaction (PCR)-based techniques to detect Mycobacterium tuberculosis DNA in respiratory specimens have been increasingly used to diagnose pulmonary tuberculosis. Their use in non-respiratory specimens to diagnose extrapulmonary tuberculosis is, however, controversial. In this study, we estimated the accuracy of three in-country commercialized PCR-based diagnostic techniques in pleural fluid samples for the diagnosis of pleural tuberculosis. Patients underwent thoracenthesis for diagnosis purposes; pleural fluid aliquots were frozen and subsequently submitted to two real time PCR tests (COBAS(®)TAQMAN(®)MTB and Xpert(®)MTB/Rif) and one conventional PCR test (Detect-TB(®)). Two different reference standards were considered: probable tuberculosis (based on clinical grounds) and confirmed tuberculosis (bacteriologically or histologically). Ninety-three patients were included, of whom 65 with pleural tuberculosis, 35 of them confirmed. Sensitivities were 29% for COBAS(®)TAQMAN(®)MTB, 3% for Xpert(®)MTB/Rif and 3% for Detect-TB(®); specificities were 86%, 100% and 97% respectively, considering confirmed tuberculosis. Considering all cases, sensitivities were 16%, 3% and 2%, and specificities, 86%, 100%, and 97%. Compared to the 95% sensitivity of adenosine deaminase, the most sensitive test for pleural tuberculosis, the sensitivities of the three PCR-based tests were very low. We conclude that at present, there is no major place for such tests in routine clinical use. Copyright © 2014 Elsevier Ltd. All rights reserved.

Differential Diagnosis of Malaria on Truelab Uno®, a Portable, Real-Time, MicroPCR Device for Point-Of-Care Applications.

PubMed

Nair, Chandrasekhar Bhaskaran; Manjula, Jagannath; Subramani, Pradeep Annamalai; Nagendrappa, Prakash B; Manoj, Mulakkapurath Narayanan; Malpani, Sukriti; Pullela, Phani Kumar; Subbarao, Pillarisetti Venkata; Ramamoorthy, Siva; Ghosh, Susanta K

2016-01-01

Sensitive and specific detection of malarial parasites is crucial in controlling the significant malaria burden in the developing world. Also important is being able to identify life threatening Plasmodium falciparum malaria quickly and accurately to reduce malaria related mortality. Existing methods such as microscopy and rapid diagnostic tests (RDTs) have major shortcomings. Here, we describe a new real-time PCR-based diagnostic test device at point-of-care service for resource-limited settings. Truenat® Malaria, a chip-based microPCR test, was developed by bigtec Labs, Bangalore, India, for differential identification of Plasmodium falciparum and Plasmodium vivax parasites. The Truenat Malaria tests runs on bigtec's Truelab Uno® microPCR device, a handheld, battery operated, and easy-to-use real-time microPCR device. The performance of Truenat® Malaria was evaluated versus the WHO nested PCR protocol. The Truenat® Malaria was further evaluated in a triple-blinded study design using a sample panel of 281 specimens created from the clinical samples characterized by expert microscopy and a rapid diagnostic test kit by the National Institute of Malaria Research (NIMR). A comparative evaluation was done on the Truelab Uno® and a commercial real-time PCR system. The limit of detection of the Truenat Malaria assay was found to be <5 parasites/μl for both P. falciparum and P. vivax. The Truenat® Malaria test was found to have sensitivity and specificity of 100% each, compared to the WHO nested PCR protocol based on the evaluation of 100 samples. The sensitivity using expert microscopy as the reference standard was determined to be around 99.3% (95% CI: 95.5-99.9) at the species level. Mixed infections were identified more accurately by Truenat Malaria (32 samples identified as mixed) versus expert microscopy and RDTs which detected 4 and 5 mixed samples, respectively. The Truenat® Malaria microPCR test is a valuable diagnostic tool and implementation should be considered not only for malaria diagnosis but also for active surveillance and epidemiological intervention.

Differential Diagnosis of Malaria on Truelab Uno®, a Portable, Real-Time, MicroPCR Device for Point-Of-Care Applications

PubMed Central

Nair, Chandrasekhar Bhaskaran; Manjula, Jagannath; Subramani, Pradeep Annamalai; Nagendrappa, Prakash B.; Manoj, Mulakkapurath Narayanan; Malpani, Sukriti; Pullela, Phani Kumar; Subbarao, Pillarisetti Venkata; Ramamoorthy, Siva; Ghosh, Susanta K.

2016-01-01

Background Sensitive and specific detection of malarial parasites is crucial in controlling the significant malaria burden in the developing world. Also important is being able to identify life threatening Plasmodium falciparum malaria quickly and accurately to reduce malaria related mortality. Existing methods such as microscopy and rapid diagnostic tests (RDTs) have major shortcomings. Here, we describe a new real-time PCR-based diagnostic test device at point-of-care service for resource-limited settings. Methods Truenat® Malaria, a chip-based microPCR test, was developed by bigtec Labs, Bangalore, India, for differential identification of Plasmodium falciparum and Plasmodium vivax parasites. The Truenat Malaria tests runs on bigtec’s Truelab Uno® microPCR device, a handheld, battery operated, and easy-to-use real-time microPCR device. The performance of Truenat® Malaria was evaluated versus the WHO nested PCR protocol. The Truenat® Malaria was further evaluated in a triple-blinded study design using a sample panel of 281 specimens created from the clinical samples characterized by expert microscopy and a rapid diagnostic test kit by the National Institute of Malaria Research (NIMR). A comparative evaluation was done on the Truelab Uno® and a commercial real-time PCR system. Results The limit of detection of the Truenat Malaria assay was found to be <5 parasites/μl for both P. falciparum and P. vivax. The Truenat® Malaria test was found to have sensitivity and specificity of 100% each, compared to the WHO nested PCR protocol based on the evaluation of 100 samples. The sensitivity using expert microscopy as the reference standard was determined to be around 99.3% (95% CI: 95.5–99.9) at the species level. Mixed infections were identified more accurately by Truenat Malaria (32 samples identified as mixed) versus expert microscopy and RDTs which detected 4 and 5 mixed samples, respectively. Conclusion The Truenat® Malaria microPCR test is a valuable diagnostic tool and implementation should be considered not only for malaria diagnosis but also for active surveillance and epidemiological intervention. PMID:26784111

Performance of Traditional and Molecular Methods for Detecting Biological Agents in Drinking Water

USGS Publications Warehouse

Francy, Donna S.; Bushon, Rebecca N.; Brady, Amie M.G.; Bertke, Erin E.; Kephart, Christopher M.; Likirdopulos, Christina A.; Mailot, Brian E.; Schaefer, Frank W.; Lindquist, H.D. Alan

2009-01-01

To reduce the impact from a possible bioterrorist attack on drinking-water supplies, analytical methods are needed to rapidly detect the presence of biological agents in water. To this end, 13 drinking-water samples were collected at 9 water-treatment plants in Ohio to assess the performance of a molecular method in comparison to traditional analytical methods that take longer to perform. Two 100-liter samples were collected at each site during each sampling event; one was seeded in the laboratory with six biological agents - Bacillus anthracis (B. anthracis), Burkholderia cepacia (as a surrogate for Bu. pseudomallei), Francisella tularensis (F. tularensis), Salmonella Typhi (S. Typhi), Vibrio cholerae (V. cholerae), and Cryptospordium parvum (C. parvum). The seeded and unseeded samples were processed by ultrafiltration and analyzed by use of quantiative polymerase chain reaction (qPCR), a molecular method, and culture methods for bacterial agents or the immunomagnetic separation/fluorescent antibody (IMS/FA) method for C. parvum as traditional methods. Six replicate seeded samples were also processed and analyzed. For traditional methods, recoveries were highly variable between samples and even between some replicate samples, ranging from below detection to greater than 100 percent. Recoveries were significantly related to water pH, specific conductance, and dissolved organic carbon (DOC) for all bacteria combined by culture methods, but none of the water-quality characteristics tested were related to recoveries of C. parvum by IMS/FA. Recoveries were not determined by qPCR because of problems in quantifying organisms by qPCR in the composite seed. Instead, qPCR results were reported as detected, not detected (no qPCR signal), or +/- detected (Cycle Threshold or 'Ct' values were greater than 40). Several sample results by qPCR were omitted from the dataset because of possible problems with qPCR reagents, primers, and probes. For the remaining 14 qPCR results (including some replicate samples), F. tularensis and V. cholerae were detected in all samples after ultrafiltration, B. anthracis was detected in 13 and +/- detected in 1 sample, and C. parvum was detected in 9 and +/- detected in 4 samples. Bu. cepacia was detected in nine samples, +/- detected in two samples, and not detected in three samples (for two out of three samples not detected, a different strain was used). The qPCR assay for V. cholerae provided two false positive - but late - signals in one unseeded sample. Numbers found by qPCR after ultrafiltration were significantly or nearly significantly related to those found by traditional methods for B. anthracis, F. tularensis, and V. cholerae but not for Bu. cepacia and C. parvum. A qPCR assay for S. Typhi was not available. The qPCR method can be used to rapidly detect B. anthracis, F. tularensis, and V. cholerae with some certainty in drinking-water samples, but additional work would be needed to optimize and test qPCR for Bu. cepacia and C. parvum and establish relations to traditional methods. The specificity for the V. cholerae assay needs to be further investigated. Evidence is provided that ultrafiltration and qPCR are promising methods to rapidly detect biological agents in the Nation's drinking-water supplies and thus reduce the impact and consequences from intentional bioterrorist events. To our knowledge, this is the first study to compare the use of traditional and qPCR methods to detect biological agents in large-volume drinking-water samples.

Development and Validation of a Laboratory-Developed Multiplex Real-Time PCR Assay on the BD Max System for Detection of Herpes Simplex Virus and Varicella-Zoster Virus DNA in Various Clinical Specimens.

PubMed

Pillet, Sylvie; Verhoeven, Paul O; Epercieux, Amélie; Bourlet, Thomas; Pozzetto, Bruno

2015-06-01

A multiplex real-time PCR (quantitative PCR [qPCR]) assay detecting herpes simplex virus (HSV) and varicella-zoster virus (VZV) DNA together with an internal control was developed on the BD Max platform combining automated DNA extraction and an open amplification procedure. Its performance was compared to those of PCR assays routinely used in the laboratory, namely, a laboratory-developed test for HSV DNA on the LightCycler instrument and a test using a commercial master mix for VZV DNA on the ABI7500fast system. Using a pool of negative cerebrospinal fluid (CSF) samples spiked with either calibrated controls for HSV-1 and VZV or dilutions of a clinical strain that was previously quantified for HSV-2, the empirical limit of detection of the BD Max assay was 195.65, 91.80, and 414.07 copies/ml for HSV-1, HSV-2, and VZV, respectively. All the samples from HSV and VZV DNA quality control panels (Quality Control for Molecular Diagnostics [QCMD], 2013, Glasgow, United Kingdom) were correctly identified by the BD Max assay. From 180 clinical specimens of various origins, 2 CSF samples were found invalid by the BD Max assay due to the absence of detection of the internal control; a concordance of 100% was observed between the BD Max assay and the corresponding routine tests. The BD Max assay detected the PCR signal 3 to 4 cycles earlier than did the routine methods. With results available within 2 h on a wide range of specimens, this sensitive and fully automated PCR assay exhibited the qualities required for detecting simultaneously HSV and VZV DNA on a routine basis. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Barcode DNA length polymorphisms vs fatty acid profiling for adulteration detection in olive oil.

PubMed

Uncu, Ali Tevfik; Uncu, Ayse Ozgur; Frary, Anne; Doganlar, Sami

2017-04-15

The aim of this study was to compare the performance of a DNA-barcode assay with fatty acid profile analysis to authenticate the botanical origin of olive oil. To achieve this aim, we performed a PCR-capillary electrophoresis (PCR-CE) approach on olive oil: seed oil blends using the plastid trnL (UAA) intron barcode. In parallel to genomic analysis, we subjected the samples to gas chromatography analysis of fatty acid composition. While the PCR-CE assay proved equally efficient as gas chromatography analysis in detecting adulteration with soybean, palm, rapeseed, sunflower, sesame, cottonseed and peanut oils, it was superior to the widely utilized analytical chemistry approach in revealing the adulterant species and detecting small quantities of corn and safflower oils in olive oil. Moreover, the DNA-based test correctly identified all tested olive oil: hazelnut oil blends whereas it was not feasible to detect hazelnut oil adulteration through fatty acid profile analysis. Thus, the present research has shown the feasibility of a PCR-CE barcode assay to detect adulteration in olive oil. Copyright © 2016 Elsevier Ltd. All rights reserved.

Differentiation between Staphylococcus aureus and coagulase-negative Staphylococcus species by real-time PCR including detection of methicillin resistants in comparison to conventional microbiology testing.

PubMed

Klaschik, Sven; Lehmann, Lutz E; Steinhagen, Folkert; Book, Malte; Molitor, Ernst; Hoeft, Andreas; Stueber, Frank

2015-03-01

Staphylococcus aureus has long been recognized as a major pathogen. Methicillin-resistant strains of S. aureus (MRSA) and methicillin-resistant strains of S. epidermidis (MRSE) are among the most prevalent multiresistant pathogens worldwide, frequently causing nosocomial and community-acquired infections. In the present pilot study, we tested a polymerase chain reaction (PCR) method to quickly differentiate Staphylococci and identify the mecA gene in a clinical setting. Compared to the conventional microbiology testing the real-time PCR assay had a higher detection rate for both S. aureus and coagulase-negative Staphylococci (CoNS; 55 vs. 32 for S. aureus and 63 vs. 24 for CoNS). Hands-on time preparing DNA, carrying out the PCR, and evaluating results was less than 5 h. The assay is largely automated, easy to adapt, and has been shown to be rapid and reliable. Fast detection and differentiation of S. aureus, CoNS, and the mecA gene by means of this real-time PCR protocol may help expedite therapeutic decision-making and enable earlier adequate antibiotic treatment. © 2014 Wiley Periodicals, Inc.

Most Probable Number Rapid Viability PCR Method to Detect Viable Spores of Bacillus anthracis in Swab Samples

DOE Office of Scientific and Technical Information (OSTI.GOV)

Letant, S E; Kane, S R; Murphy, G A

2008-05-30

This note presents a comparison of Most-Probable-Number Rapid Viability (MPN-RV) PCR and traditional culture methods for the quantification of Bacillus anthracis Sterne spores in macrofoam swabs generated by the Centers for Disease Control and Prevention (CDC) for a multi-center validation study aimed at testing environmental swab processing methods for recovery, detection, and quantification of viable B. anthracis spores from surfaces. Results show that spore numbers provided by the MPN RV-PCR method were in statistical agreement with the CDC conventional culture method for all three levels of spores tested (10{sup 4}, 10{sup 2}, and 10 spores) even in the presence ofmore » dirt. In addition to detecting low levels of spores in environmental conditions, the MPN RV-PCR method is specific, and compatible with automated high-throughput sample processing and analysis protocols.« less

Influence of enrichment broths on multiplex PCR detection of total coliform bacteria, Escherichia coli and Clostridium perfringens, in spiked water samples.

PubMed

Worakhunpiset, S; Tharnpoophasiam, P

2009-07-01

Although multiplex PCR amplification condition for simultaneous detection of total coliform bacteria, Escherichia coli and Clostridium perfringens in water sample has been developed, results with high sensitivity are obtained when amplifying purified DNA, but the sensitivity is low when applied to spiked water samples. An enrichment broth culture prior PCR analysis increases sensitivity of the test but the specific nature of enrichment broth can affect the PCR results. Three enrichment broths, lactose broth, reinforced clostridial medium and fluid thioglycollate broth, were compared for their influence on sensitivity and on time required with multiplex PCR assay. Fluid thioglycollate broth was the most effective with shortest enrichment time and lowest detection limit.

Detection of Trypanosoma cruzi in untreated chronic chagasic patients is improved by using three parasitological methods simultaneously.

PubMed

Zulantay, Inés; Apt, Werner; Valencia, Claudio; Torres, Alberto; Saavedra, Miguel; Rodríguez, Jorge; Sandoval, Lea; Martínez, Gabriela; Thieme, Patricio; Sepúlveda, Eduardo

2011-10-01

This study compared three parasitological methods applied simultaneously in individuals with untreated chronic Chagas' disease in order to determine their individual and combined performances. From a total of 100 chronic chagasic patients from endemic areas of Chile, with informed consent, we extracted 2 mL of peripheral venous blood for PCR (PCR-B) and applied two xenodiagnosis (XD) boxes with seven uninfected Triatoma infestans nymphs each for microscopic examination and PCR of faecal samples of the triatomines fed on each patient (PCR-XD). The PCR-B and PCR-XD reactions were performed with oligonucleotides 121 and 122, which anneal to the four constant regions of the minicircles of Trypanosoma cruzi kinetoplasts. The 330 bp PCR product was analysed by electrophoresis in a 2% agarose gel and visualized by staining with ethidium bromide. PCR-B detected T. cruzi in 58% of the cases, while PCR-XD proved to be more sensitive than XD (67% versus 14%, respectively) (P = 0.0001). There was no difference between the detection power of PCR-B and PCR-XD (P = 0.222). The percentage detected as positive was much greater when the three tests were considered (84%) (P = 0.00001). The simultaneous application of more than one technique for the parasitological diagnosis of Chagas' disease in untreated individuals increases the possibility of detection of T. cruzi.

Bayesian modelling to estimate the test characteristics of coprology, coproantigen ELISA and a novel real-time PCR for the diagnosis of taeniasis.

PubMed

Praet, Nicolas; Verweij, Jaco J; Mwape, Kabemba E; Phiri, Isaac K; Muma, John B; Zulu, Gideon; van Lieshout, Lisette; Rodriguez-Hidalgo, Richar; Benitez-Ortiz, Washington; Dorny, Pierre; Gabriël, Sarah

2013-05-01

To estimate and compare the performances of coprology, copro-Ag ELISA and real-time polymerase chain reaction assay (copro-PCR) for detection of Taenia solium tapeworm carriers. The three diagnostic tests were applied on 817 stool samples collected in two Zambian communities where taeniasis is endemic. A Bayesian approach was used to allow estimation of the test characteristics. Two (0.2%; 95% Confidence Interval (CI): 0-0.8), 67 (8.2%; 95% CI: 6.4-10.3) and 10 (1.2%; 95% CI: 0.5-2.2) samples were positive using coprology, copro-Ag ELISA and copro-PCR, respectively. Specificities of 99.9%, 92.0% and 99.0% were determined for coprology, copro-Ag ELISA and copro-PCR, respectively. Sensitivities of 52.5%, 84.5% and 82.7% were determined for coprology, copro-Ag ELISA and copro-PCR, respectively. We urge for additional studies exploring possible cross-reactions of the copro-Ag ELISA and for the use of more sensitive tests, such as copro-PCR, for the detection of tapeworm carriers, which is a key factor in controlling the parasite in endemic areas. © 2013 Blackwell Publishing Ltd.

Leishmania Infection: Laboratory Diagnosing in the Absence of a “Gold Standard”

PubMed Central

Rodríguez-Cortés, Alhelí; Ojeda, Ana; Francino, Olga; López-Fuertes, Laura; Timón, Marcos; Alberola, Jordi

2010-01-01

There is no gold standard for diagnosing leishmaniases. Our aim was to assess the operative validity of tests used in detecting Leishmania infection using samples from experimental infections, a reliable equivalent to the classic definition of gold standard. Without statistical differences, the highest sensitivity was achieved by protein A (ProtA), immunoglobulin (Ig)G2, indirect fluorescenece antibody test (IFAT), lymphocyte proliferation assay, quantitative real-time polymerase chain reaction of bone marrow (qPCR-BM), qPCR-Blood, and IgG; and the highest specificity by IgG1, IgM, IgA, qPCR-Blood, IgG, IgG2, and qPCR-BM. Maximum positive predictive value was obtained simultaneously by IgG2, qPCR-Blood, and IgG; and maximum negative predictive value by qPCR-BM. Best positive and negative likelihood ratios were obtained by IgG2. The test having the greatest, statistically significant, area under the receiver operating characteristics curve was IgG2 enzyme-linked immunosorbent assay (ELISA). Thus, according to the gold standard used, IFAT and qPCR are far from fulfilling the requirements to be considered gold standards, and the test showing the highest potential to detect Leishmania infection is Leishmania-specific ELISA IgG2. PMID:20134001

Leishmania infection: laboratory diagnosing in the absence of a "gold standard".

PubMed

Rodríguez-Cortés, Alhelí; Ojeda, Ana; Francino, Olga; López-Fuertes, Laura; Timón, Marcos; Alberola, Jordi

2010-02-01

There is no gold standard for diagnosing leishmaniases. Our aim was to assess the operative validity of tests used in detecting Leishmania infection using samples from experimental infections, a reliable equivalent to the classic definition of gold standard. Without statistical differences, the highest sensitivity was achieved by protein A (ProtA), immunoglobulin (Ig)G2, indirect fluorescenece antibody test (IFAT), lymphocyte proliferation assay, quantitative real-time polymerase chain reaction of bone marrow (qPCR-BM), qPCR-Blood, and IgG; and the highest specificity by IgG1, IgM, IgA, qPCR-Blood, IgG, IgG2, and qPCR-BM. Maximum positive predictive value was obtained simultaneously by IgG2, qPCR-Blood, and IgG; and maximum negative predictive value by qPCR-BM. Best positive and negative likelihood ratios were obtained by IgG2. The test having the greatest, statistically significant, area under the receiver operating characteristics curve was IgG2 enzyme-linked immunosorbent assay (ELISA). Thus, according to the gold standard used, IFAT and qPCR are far from fulfilling the requirements to be considered gold standards, and the test showing the highest potential to detect Leishmania infection is Leishmania-specific ELISA IgG2.

Rapid detection of coliforms in drinking water of Arak city using multiplex PCR method in comparison with the standard method of culture (Most Probably Number)

PubMed Central

Fatemeh, Dehghan; Reza, Zolfaghari Mohammad; Mohammad, Arjomandzadegan; Salomeh, Kalantari; Reza, Ahmari Gholam; Hossein, Sarmadian; Maryam, Sadrnia; Azam, Ahmadi; Mana, Shojapoor; Negin, Najarian; Reza, Kasravi Alii; Saeed, Falahat

2014-01-01

Objective To analyse molecular detection of coliforms and shorten the time of PCR. Methods Rapid detection of coliforms by amplification of lacZ and uidA genes in a multiplex PCR reaction was designed and performed in comparison with most probably number (MPN) method for 16 artificial and 101 field samples. The molecular method was also conducted on isolated coliforms from positive MPN samples; standard sample for verification of microbial method certificated reference material; isolated strains from certificated reference material and standard bacteria. The PCR and electrophoresis parameters were changed for reducing the operation time. Results Results of PCR for lacZ and uidA genes were similar in all of standard, operational and artificial samples and showed the 876 bp and 147 bp bands of lacZ and uidA genes by multiplex PCR. PCR results were confirmed by MPN culture method by sensitivity 86% (95% CI: 0.71-0.93). Also the total execution time, with a successful change of factors, was reduced to less than two and a half hour. Conclusions Multiplex PCR method with shortened operation time was used for the simultaneous detection of total coliforms and Escherichia coli in distribution system of Arak city. It's recommended to be used at least as an initial screening test, and then the positive samples could be randomly tested by MPN. PMID:25182727

PCR diagnostics underestimate the prevalence of avian malaria (Plasmodium relictum) in experimentally-infected passerines

USGS Publications Warehouse

Jarvi, Susan I.; Schultz, Jeffrey J.; Atkinson, Carter T.

2002-01-01

Several polymerase chain reaction (PCR)-based methods have recently been developed for diagnosing malarial infections in both birds and reptiles, but a critical evaluation of their sensitivity in experimentally-infected hosts has not been done. This study compares the sensitivity of several PCR-based methods for diagnosing avian malaria (Plasmodium relictum) in captive Hawaiian honeycreepers using microscopy and a recently developed immunoblotting technique. Sequential blood samples were collected over periods of up to 4.4 yr after experimental infection and rechallenge to determine both the duration and detectability of chronic infections. Two new nested PCR approaches for detecting circulating parasites based on P. relictum 18S rRNA genes and the thrombospondin-related anonymous protein (TRAP) gene are described. The blood smear and the PCR tests were less sensitive than serological methods for detecting chronic malarial infections. Individually, none of the diagnostic methods was 100% accurate in detecting subpatent infections, although serological methods were significantly more sensitive (97%) than either nested PCR (61–84%) or microscopy (27%). Circulating parasites in chronically infected birds either disappear completely from circulation or to drop to intensities below detectability by nested PCR. Thus, the use of PCR as a sole means of detection of circulating parasites may significantly underestimate true prevalence.

Diagnostic accuracy of two multiplex real-time polymerase chain reaction assays for the diagnosis of meningitis in children in a resource-limited setting.

PubMed

Khumalo, Jermaine; Nicol, Mark; Hardie, Diana; Muloiwa, Rudzani; Mteshana, Phindile; Bamford, Colleen

2017-01-01

Accurate etiological diagnosis of meningitis is important, but difficult in resource-limited settings due to prior administration of antibiotics and lack of viral diagnostics. We aimed to develop and validate 2 real-time multiplex PCR (RT-PCR) assays for the detection of common causes of community-acquired bacterial and viral meningitis in South African children. We developed 2 multiplex RT- PCRs for detection of S. pneumoniae, N. meningitidis, H. influenzae, enteroviruses, mumps virus and herpes simplex virus. We tested residual CSF samples from children presenting to a local paediatric hospital over a one-year period, whose CSF showed an abnormal cell count. Results were compared with routine diagnostic tests and the final discharge diagnosis. We calculated accuracy of the bacterial RT-PCR assay compared to CSF culture and using World Health Organisation definitions of laboratory-confirmed bacterial meningitis. From 292 samples, bacterial DNA was detected in 12 (4.1%) and viral nucleic acids in 94 (32%). Compared to CSF culture, the sensitivity and specificity of the bacterial RT-PCR was 100% and 97.2% with complete agreement in organism identification. None of the cases positive by viral RT-PCR had a bacterial cause confirmed on CSF culture. Only 9/90 (10%) of patients diagnosed clinically as bacterial meningitis or partially treated bacterial meningitis tested positive with the bacterial RT-PCR. In this population the use of 2 multiplex RT-PCRs targeting 6 common pathogens gave promising results. If introduced into routine diagnostic testing, these multiplex RT-PCR assays would supplement other diagnostic tests, and have the potential to limit unnecessary antibiotic therapy and hospitalisation.

Diagnostic accuracy of two multiplex real-time polymerase chain reaction assays for the diagnosis of meningitis in children in a resource-limited setting

PubMed Central

Khumalo, Jermaine; Nicol, Mark; Hardie, Diana; Muloiwa, Rudzani; Mteshana, Phindile

2017-01-01

Introduction Accurate etiological diagnosis of meningitis is important, but difficult in resource-limited settings due to prior administration of antibiotics and lack of viral diagnostics. We aimed to develop and validate 2 real-time multiplex PCR (RT-PCR) assays for the detection of common causes of community-acquired bacterial and viral meningitis in South African children. Methods We developed 2 multiplex RT- PCRs for detection of S. pneumoniae, N. meningitidis, H. influenzae, enteroviruses, mumps virus and herpes simplex virus. We tested residual CSF samples from children presenting to a local paediatric hospital over a one-year period, whose CSF showed an abnormal cell count. Results were compared with routine diagnostic tests and the final discharge diagnosis. We calculated accuracy of the bacterial RT-PCR assay compared to CSF culture and using World Health Organisation definitions of laboratory-confirmed bacterial meningitis. Results From 292 samples, bacterial DNA was detected in 12 (4.1%) and viral nucleic acids in 94 (32%). Compared to CSF culture, the sensitivity and specificity of the bacterial RT-PCR was 100% and 97.2% with complete agreement in organism identification. None of the cases positive by viral RT-PCR had a bacterial cause confirmed on CSF culture. Only 9/90 (10%) of patients diagnosed clinically as bacterial meningitis or partially treated bacterial meningitis tested positive with the bacterial RT-PCR. Discussion In this population the use of 2 multiplex RT-PCRs targeting 6 common pathogens gave promising results. If introduced into routine diagnostic testing, these multiplex RT-PCR assays would supplement other diagnostic tests, and have the potential to limit unnecessary antibiotic therapy and hospitalisation. PMID:28346504

Evaluation of conventional PCR for detection of Strongylus vulgaris on horse farms.

PubMed

Bracken, M K; Wøhlk, C B M; Petersen, S L; Nielsen, M K

2012-03-23

Strongyle parasites are ubiquitous in grazing horses. Of these, the bloodworm Strongylus vulgaris is regarded as most pathogenic. Increasing levels of anthelmintic resistance in strongyle parasites has led to recommendations of decreased treatment intensities, and there is now a pronounced need for reliable tools for detection of parasite burdens in general and S. vulgaris in particular. The only method currently available for diagnosing S. vulgaris in practice is the larval culture, which is laborious and time-consuming, so veterinary practitioners most often pool samples from several horses together in one culture to save time. Recently, molecular tools have been developed to detect S. vulgaris in faecal samples. The aim of this study was to compare the performance of a conventional polymerase chain reaction (PCR) assay with the traditional larval culture and furthermore test the performance of pooled versus individual PCR for farm screening purposes. Faecal samples were obtained from 331 horses on 18 different farms. Farm size ranged from 6 to 56 horses, and horses aged between 2 months and 31 years. Larval cultures and PCR were performed individually on all horses. In addition, PCR was performed on 66 faecal pools consisting of 3-5 horses each. Species-specific PCR primers previously developed were used for the PCR. PCR and larval culture detected S. vulgaris in 12.1 and 4.5% of individual horses, respectively. On the farm level, eight farms tested positive with the larval culture, while 13 and 11 farms were positive with the individual and pooled PCRs, respectively. The individual PCR method was statistically superior to the larval culture, while no statistical difference could be detected between pooled and individual PCR for farm screening. In conclusion, pooled PCR appears to be a useful tool for farm screening for S. vulgaris. Copyright © 2011 Elsevier B.V. All rights reserved.

Molecular diagnosis of Plasmodium ovale by photo-induced electron transfer fluorogenic primers: PET-PCR

PubMed Central

Akerele, David; Ljolje, Dragan; Talundzic, Eldin; Udhayakumar, Venkatachalam

2017-01-01

Accurate diagnosis of malaria infections continues to be challenging and elusive, especially in the detection of submicroscopic infections. Developing new malaria diagnostic tools that are sensitive enough to detect low-level infections, user friendly, cost effective and capable of performing large scale diagnosis, remains critical. We have designed novel self-quenching photo-induced electron transfer (PET) fluorogenic primers for the detection of P. ovale by real-time PCR. In our study, a total of 173 clinical samples, consisting of different malaria species, were utilized to test this novel PET-PCR primer. The sensitivity and specificity were calculated using nested-PCR as the reference test. The novel primer set demonstrated a sensitivity of 97.5% and a specificity of 99.2% (95% CI 85.2–99.8% and 95.2–99.9% respectively). Furthermore, the limit of detection for P. ovale was found to be 1 parasite/μl. The PET-PCR assay is a new molecular diagnostic tool with comparable performance to other commonly used PCR methods. It is relatively easy to perform, and amiable to large scale malaria surveillance studies and malaria control and elimination programs. Further field validation of this novel primer will be helpful to ascertain the utility for large scale malaria screening programs. PMID:28640824

Molecular diagnosis of Plasmodium ovale by photo-induced electron transfer fluorogenic primers: PET-PCR.

PubMed

Akerele, David; Ljolje, Dragan; Talundzic, Eldin; Udhayakumar, Venkatachalam; Lucchi, Naomi W

2017-01-01

Accurate diagnosis of malaria infections continues to be challenging and elusive, especially in the detection of submicroscopic infections. Developing new malaria diagnostic tools that are sensitive enough to detect low-level infections, user friendly, cost effective and capable of performing large scale diagnosis, remains critical. We have designed novel self-quenching photo-induced electron transfer (PET) fluorogenic primers for the detection of P. ovale by real-time PCR. In our study, a total of 173 clinical samples, consisting of different malaria species, were utilized to test this novel PET-PCR primer. The sensitivity and specificity were calculated using nested-PCR as the reference test. The novel primer set demonstrated a sensitivity of 97.5% and a specificity of 99.2% (95% CI 85.2-99.8% and 95.2-99.9% respectively). Furthermore, the limit of detection for P. ovale was found to be 1 parasite/μl. The PET-PCR assay is a new molecular diagnostic tool with comparable performance to other commonly used PCR methods. It is relatively easy to perform, and amiable to large scale malaria surveillance studies and malaria control and elimination programs. Further field validation of this novel primer will be helpful to ascertain the utility for large scale malaria screening programs.

Multiplex PCR detection of waterborne intestinal protozoa: microsporidia, Cyclospora, and Cryptosporidium.

PubMed

Lee, Seung-Hyun; Joung, Migyo; Yoon, Sejoung; Choi, Kyoungjin; Park, Woo-Yoon; Yu, Jae-Ran

2010-12-01

Recently, emerging waterborne protozoa, such as microsporidia, Cyclospora, and Cryptosporidium, have become a challenge to human health worldwide. Rapid, simple, and economical detection methods for these major waterborne protozoa in environmental and clinical samples are necessary to control infection and improve public health. In the present study, we developed a multiplex PCR test that is able to detect all these 3 major waterborne protozoa at the same time. Detection limits of the multiplex PCR method ranged from 10(1) to 10(2) oocysts or spores. The primers for microsporidia or Cryptosporidium used in this study can detect both Enterocytozoon bieneusi and Encephalitozoon intestinalis, or both Cryptosporidium hominis and Cryptosporidium parvum, respectively. Restriction enzyme digestion of PCR products with BsaBI or BsiEI makes it possible to distinguish the 2 species of microsporidia or Cryptosporidium, respectively. This simple, rapid, and cost-effective multiplex PCR method will be useful for detecting outbreaks or sporadic cases of waterborne protozoa infections.

The usefulness of direct agglutination test, enzyme-linked immunosorbent assay and polymerase chain reaction for the detection of Toxoplasma gondii in wild animals.

PubMed

Kornacka, Aleksandra; Cybulska, Aleksandra; Bień, Justyna; Goździk, Katarzyna; Moskwa, Bożena

2016-09-15

The aim of the study was to compare the usefulness of two antibody-based methods, the direct agglutination test (DAT) and enzyme linked immuosorbent assay (ELISA), with that of the polymerase chain reaction (PCR) for detecting anti-Toxoplasma gondii in samples derived from naturally-infected wild animals. Antibodies against T. gondii were detected in meat juice samples collected from 129 free- living carnivores and omnivores. T. gondii seroprevalence was confirmed in 73,6% of examined samples when DAT and ELISA were used separately, but in only 88,4% samples when both immunological tests were used in parallel. PCR results confirmed the presence of DNA of the parasite in 24 of all the 129 samples. Sixteen samples were classified as positive when all three tests were used. A moderate degree of agreement was found between DAT and ELISA (κ=0.55). However, no agreement was found between the molecular and serological tests: κ=-1.75 for DAT versus PCR; κ=-1.67 ELISA versus PCR. By using both serological tests, antibodies against T. gondii were found in 77.5% of red foxes, 12.5% of badgers, 40% of martens and 8.3% of raccoon dogs. Antibodies against the parasite were detected also in one mink, but not in the sample derived from a polecat. T.gondii DNA was found in the brain tissue of 20 red foxes, three badgers and one raccoon dog. Our studies confirm that ELISA and DAT are suitable and reliable techniques for T. gondii antibody detection in meat juice from wild animals when serum samples are unavailable. Positive results obtained by immunological tests do not always reflect that the host was infected by T. gondii. They indicate only a contact with parasite. PCR should be used to confirm te presence of DNA from T. gondii. Copyright © 2016 Elsevier B.V. All rights reserved.

Evaluation of PCR Systems for Field Screening of Bacillus anthracis

PubMed Central

Ozanich, Richard M.; Colburn, Heather A.; Victry, Kristin D.; Bartholomew, Rachel A.; Arce, Jennifer S.; Heredia-Langner, Alejandro; Jarman, Kristin; Kreuzer, Helen W.

2017-01-01

There is little published data on the performance of hand-portable polymerase chain reaction (PCR) systems that can be used by first responders to determine if a suspicious powder contains a potential biothreat agent. We evaluated 5 commercially available hand-portable PCR instruments for detection of Bacillus anthracis. We used a cost-effective, statistically based test plan to evaluate systems at performance levels ranging from 0.85-0.95 lower confidence bound (LCB) of the probability of detection (POD) at confidence levels of 80% to 95%. We assessed specificity using purified genomic DNA from 13 B. anthracis strains and 18 Bacillus near neighbors, potential interference with 22 suspicious powders that are commonly encountered in the field by first responders during suspected biothreat incidents, and the potential for PCR inhibition when B. anthracis spores were spiked into these powders. Our results indicate that 3 of the 5 systems achieved 0.95 LCB of the probability of detection with 95% confidence levels at test concentrations of 2,000 genome equivalents/mL (GE/mL), which is comparable to 2,000 spores/mL. This is more than sufficient sensitivity for screening visible suspicious powders. These systems exhibited no false-positive results or PCR inhibition with common suspicious powders and reliably detected B. anthracis spores spiked into these powders, though some issues with assay controls were observed. Our testing approach enables efficient performance testing using a statistically rigorous and cost-effective test plan to generate performance data that allow users to make informed decisions regarding the purchase and use of field biodetection equipment. PMID:28192050

Single-Reaction Multiplex Reverse Transcription PCR for Detection of Zika, Chikungunya, and Dengue Viruses

PubMed Central

Waggoner, Jesse J.; Gresh, Lionel; Mohamed-Hadley, Alisha; Ballesteros, Gabriela; Davila, Maria Jose Vargas; Tellez, Yolanda; Sahoo, Malaya K.; Balmaseda, Angel; Harris, Eva

2016-01-01

Clinical manifestations of Zika virus, chikungunya virus, and dengue virus infections can be similar. To improve virus detection, streamline molecular workflow, and decrease test costs, we developed and evaluated a multiplex real-time reverse transcription PCR for these viruses. PMID:27184629

Reduced-cost Chlamydia trachomatis-specific multiplex real-time PCR diagnostic assay evaluated for ocular swabs and use by trachoma research programmes.

PubMed

Butcher, Robert; Houghton, Jo; Derrick, Tamsyn; Ramadhani, Athumani; Herrera, Beatriz; Last, Anna R; Massae, Patrick A; Burton, Matthew J; Holland, Martin J; Roberts, Chrissy H

2017-08-01

Trachoma, caused by the intracellular bacterium Chlamydia trachomatis (Ct), is the leading infectious cause of preventable blindness. Many commercial platforms are available that provide highly sensitive and specific detection of Ct DNA. However, the majority of these commercial platforms are inaccessible for population-level surveys in resource-limited settings typical to trachoma control programmes. We developed two low-cost quantitative PCR (qPCR) tests for Ct using readily available reagents on standard real-time thermocyclers. Each multiplex qPCR test targets one genomic and one plasmid Ct target in addition to an endogenous positive control for Homo sapiens DNA. The quantitative performance of the qPCR assays in clinical samples was determined by comparison to a previously evaluated droplet digital PCR (ddPCR) test. The diagnostic performance of the qPCR assays were evaluated against a commercial assay (artus C. trachomatis Plus RG PCR, Qiagen) using molecular diagnostics quality control standards and clinical samples. We examined the yield of Ct DNA prepared from five different DNA extraction kits and a cold chain-free dry-sample preservation method using swabs spiked with fixed concentrations of human and Ct DNA. The qPCR assay was highly reproducible (Ct plasmid and genomic targets mean total coefficients of variance 41.5% and 48.3%, respectively). The assay detected 8/8 core specimens upon testing of a quality control panel and performed well in comparison to commercially marketed comparator test (sensitivity and specificity>90%). Optimal extraction and sample preservation methods for research applications were identified. We describe a pipeline from collection to diagnosis providing the most efficient sample preservation and extraction with significant per test cost savings over a commercial qPCR diagnostic assay. The assay and its evaluation should allow control programs wishing to conduct independent research within the context of trachoma control, access to an affordable test with defined performance characteristics. Copyright © 2017. Published by Elsevier B.V.

Rapid diagnosis of sepsis with TaqMan-Based multiplex real-time PCR.

PubMed

Liu, Chang-Feng; Shi, Xin-Ping; Chen, Yun; Jin, Ye; Zhang, Bing

2018-02-01

The survival rate of septic patients mainly depends on a rapid and reliable diagnosis. A rapid, broad range, specific and sensitive quantitative diagnostic test is the urgent need. Thus, we developed a TaqMan-Based Multiplex real-time PCR assays to identify bloodstream pathogens within a few hours. Primers and TaqMan probes were designed to be complementary to conserved regions in the 16S rDNA gene of different kinds of bacteria. To evaluate accurately, sensitively, and specifically, the known bacteria samples (Standard strains, whole blood samples) are determined by TaqMan-Based Multiplex real-time PCR. In addition, 30 blood samples taken from patients with clinical symptoms of sepsis were tested by TaqMan-Based Multiplex real-time PCR and blood culture. The mean frequency of positive for Multiplex real-time PCR was 96% at a concentration of 100 CFU/mL, and it was 100% at a concentration greater than 1000 CFU/mL. All the known blood samples and Standard strains were detected positively by TaqMan-Based Multiplex PCR, no PCR products were detected when DNAs from other bacterium were used in the multiplex assay. Among the 30 patients with clinical symptoms of sepsis, 18 patients were confirmed positive by Multiplex real-time PCR and seven patients were confirmed positive by blood culture. TaqMan-Based Multiplex real-time PCR assay with highly sensitivity, specificity and broad detection range, is a rapid and accurate method in the detection of bacterial pathogens of sepsis and should have a promising usage in the diagnosis of sepsis. © 2017 Wiley Periodicals, Inc.

Evaluation of two singleplex reverse transcription-Insulated isothermal PCR tests and a duplex real-time RT-PCR test for the detection of porcine epidemic diarrhea virus and porcine deltacoronavirus.

PubMed

Zhang, Jianqiang; Tsai, Yun-Long; Lee, Pei-Yu Alison; Chen, Qi; Zhang, Yan; Chiang, Cheng-Jen; Shen, Yu-Han; Li, Fu-Chun; Chang, Hsiao-Fen Grace; Gauger, Phillip C; Harmon, Karen M; Wang, Hwa-Tang Thomas

2016-08-01

Recent outbreaks of porcine epidemic diarrhea virus (PEDV) and porcine deltacoronavirus (PDCoV) in multiple countries have caused significant economic losses and remain a serious challenge to the swine industry. Rapid diagnosis is critical for the implementation of efficient control strategies before and during PEDV and PDCoV outbreaks. Insulated isothermal PCR (iiPCR) on the portable POCKIT™ device is user friendly for on-site pathogen detection. In the present study, a singleplex PEDV RT-iiPCR, a singleplex PDCoV RT-iiPCR, and a duplex PEDV/PDCoV real-time RT-PCR (rRT-PCR) commercial reagents targeting the M gene were compared to an N gene-based PEDV rRT-PCR and an M gene-based PDCoV rRT-PCR that were previously published and used as reference PCRs. All PCR assays were highly specific and did not cross react with other porcine enteric pathogens. Analytical sensitivities of the PEDV RT-iiPCR, PDCoV RT-iiPCR and duplex PEDV/PDCoV rRT-PCR were determined using in vitro transcribed RNA as well as viral RNA extracted from ten-fold serial dilutions of PEDV and PDCoV cell culture isolates. Performance of each PCR assay was further evaluated using 170 clinical samples (86 fecal swabs, 24 feces, 19 intestines, and 41 oral fluids). Compared to the reference PEDV rRT-PCR, the sensitivity, specificity and accuracy of the PEDV RT-iiPCR were 97.73%, 98.78%, and 98.24%, respectively, and those of the duplex PEDV/PDCoV rRT-PCR were 98.86%, 96.34%, and 97.65%, respectively. Compared to the reference PDCoV rRT-PCR, the sensitivity, specificity and accuracy of the PDCoV RT-iiPCR were 100%, 100%, and 100%, respectively, and those of the PEDV/PDCoV duplex rRT-PCR were 96.34%, 100%, and 98.24%, respectively. Overall, all three new PCR assays were comparable to the reference rRT-PCRs for detection of PEDV and/or PDCoV. The PEDV and PDCoV RT-iiPCRs are potentially useful tools for on-site detection and the duplex PEDV/PDCoV rRT-PCR provides a convenient method to simultaneously detect the two viruses and differentiate PEDV from PDCoV. Copyright © 2016 Elsevier B.V. All rights reserved.

A novel multiplex real-time PCR assay for the concurrent detection of hepatitis A, B and C viruses in patients with acute hepatitis.

PubMed

Park, Yongjung; Kim, Beom Seok; Choi, Kyu Hun; Shin, Dong Ho; Lee, Mi Jung; Cho, Yonggeun; Kim, Hyon-Suk

2012-01-01

A novel multiplex real-time PCR assay for concurrent detection of hepatitis viruses was evaluated for its clinical performance in screening patients with acute hepatitis. A total of 648 serum samples were collected from patients with acute symptoms of hepatitis. Concurrent detection of nucleic acids of HAV, HBV and HCV was performed using the Magicplex™ HepaTrio Real-time Detection test. Serum nucleic acid levels of HBV and HCV were also quantified by the Cobas® AmpliPrep/Cobas® TaqMan® (CAP/CTM) HBV and HCV tests. Patients' medical records were also reviewed. Concordance rates between the results from the HepaTrio and the CAP/CTM tests for the detection of HBV and HCV were 94.9% (k = 0.88) and 99.2% (k = 0.98), respectively. The cycle threshold values with the HepaTrio test were also correlated well with the levels of HBV DNA (r = -0.9230) and HCV RNA (r = -0.8458). The sensitivity and specificity of the HepaTrio test were 93.8% and 98.2%, respectively, for detecting HBV infection, and 99.1% and 100.0%, respectively, for HCV infection. For the HepaTrio test, 21 (3.2%) cases were positive for both HBV and HCV. Among the positive cases, 6 (0.9%) were true coinfections. This test also detected 18 (2.8%) HAV positives. The HepaTrio test demonstrated good clinical performance and produced results that agreed well with those of the CAP/CTM assays, especially for the detection of HCV. This assay was also able to detect HAV RNA from anti-HAV IgM-positive individuals. Therefore, this new multiplex PCR assay could be useful for the concurrent detection of the three hepatitis viruses.

Enhanced Reverse Transcription-PCR Assay for Detection of Norovirus Genogroup I

PubMed Central

Dreier, Jens; Störmer, Melanie; Mäde, Dietrich; Burkhardt, Sabine; Kleesiek, Knut

2006-01-01

We have developed a one-tube reverse transcription (RT)-PCR method using the real-time TaqMan PCR system for the detection of norovirus genogroup I (NV GGI). By introduction of a novel probe based on locked nucleic acid technology, we enhanced the sensitivity of the assay compared to those of conventional TaqMan probes. The sensitivity of the NV GGI RT-PCR was determined by probit analysis with defined RNA standards and quantified norovirus isolates to 711 copies/ml (95% detection limit). In order to detect PCR inhibition, we included a heterologous internal control (IC) system based on phage MS2. This internally controlled RT-PCR was tested on different real-time PCR platforms, LightCycler, Rotorgene, Mastercycler EP realplex, and ABI Prism. Compared to the assay without an IC, the duplex RT-PCR exhibited no reduction in sensitivity in clinical samples. In combination with an established NV GGII real-time RT-PCR, we used the novel assay in a routine assay for diagnosis of clinical and food-borne norovirus infection. We applied this novel assay to analyze outbreaks of nonbacterial acute gastroenteritis. Norovirus of GGI was detected in these outbreaks. Sequence and similarity plot analysis of open reading frame 1 (ORF1) and ORF2 showed two genotypes, GGI/2 and GGI/4, in semiclosed communities. PMID:16891482

Fluorescence in situ hybridization is superior for monitoring Epstein Barr viral load in infectious mononucleosis patients.

PubMed

Cao, Pengfei; Zhang, Meili; Wang, Wei; Dai, Yafei; Sai, Buqing; Sun, Jun; Wang, Lujuan; Wang, Fan; Li, Guiyuan; Xiang, Juanjuan

2017-05-03

Epstein Barr virus (EBV) plays a causal role in some diseases, including infectious mononucleosis, lymphoproliferative diseases and nasopharyngeal carcinoma. Detection of EBV infection has been shown to be a useful tool for diagnosing EBV-related diseases. In the present study, we compared the performance of molecular tests, including fluorescence in situ hybridization (FISH) and EBV real-time PCR, to those of serological assays for the detection of EBV infection. Thirty-eight patients with infectious mononucleosis (IM) were enrolled, of whom 31 were diagnosed with a mild type, and seven were diagnosed with IM with haemophagocytic lymphohistiocytosis and chronic active EBV infection. Twenty healthy controls were involved in the study. The atypical lymphocytes in peripheral blood were detected under a microscope and the percentage of positive cells was calculated. EBV DNA load in peripheral blood was detected using real-time PCR. The FISH assay was developed to detect the EBV genome from peripheral blood mononuclear cells (PBMC). Other diagnosis methods including the heterophil agglutination (HA) test and EBV-VCA-IgM test, to detect EBV were also compared. SPSS17.0 was used for statistical analysis. In all, 5-41% atypical lymphocytes were found among the PBMC in mild IM patients, whereas 8-51% atypical lymphocytes were found in IM patients with haemophagocytic lymphohistiocytosis and chronic active EBV infection patients. There was no significant difference in the ratios of atypical lymphoma between patients of the different types. We observed that 71.2% of mild IM patients and 85.7% of IM patients with haemophagocytic lymphohistiocytosis and chronic active EBV infection patients were positive for EBV-VCA-IgM. EBV-VCA-IgM was negative in all healthy control subjects. In addition, 67.1% of mild IM patients tested heterophile antibody positive, whereas 71.4% of IM patients with haemophagocytic lymphohistiocytosis and chronic active EBV infection tested positive. EBV DNA detected using real-time PCR was observed in 89.5% of these IM patients. The EBV genome was detected by the FISH assay in 97.4% of the IM patients. The EB viral loads detected by FISH and real-time PCR increased with the severity of IM. The EBV genome was detected in almost all the PBMC of IM with haemophagocytic lymphohistiocytosis and chronic active EBV infection patients. Molecular tests, including FISH and EBV real-time PCR, are more sensitive than serological assays for the detection of EBV infection. The FISH assay detecting EBV copies in unfractionated whole blood is preferable and superior to plasma real-time PCR in its reflection of the absolute viral burden circulating in the patients.

Detection of reticuloendotheliosis virus as a contaminant of fowl pox vaccines.

PubMed

Awad, A M; Abd El-Hamid, H S; Abou Rawash, A A; Ibrahim, H H

2010-11-01

This study was designed to detect reticuloendotheliosis virus (REV) as a contaminant in fowl pox vaccines. A total of 30 fowl pox vaccine samples were examined for the presence of REV using both in vitro and in vivo methods. In in vitro testing, the fowl pox vaccine samples were inoculated into chicken embryo fibroblast cultures prepared from specific-pathogen-free embryonated chicken eggs, and the cultures were examined using PCR to detect REV. In in vivo testing, each fowl pox vaccine sample was inoculated into 5-d-old specific-pathogen-free chicks, which were kept under observation for up to 12 wk postinoculation; serum samples were collected at 15, 30, and 45 d postinoculation for the detection of REV-specific antibodies using ELISA. Tissue samples were collected at 8 and 12 wk postinoculation for histopathological examination. Of the tested vaccines, only one imported vaccine sample tested positive for REV using PCR. Serum samples collected from chicks infected with the PCR-positive vaccine batch also tested positive for REV-specific antibodies using ELISA. Histopathological examination of the liver, spleen, and bursa of Fabricius demonstrated the presence of tumor cells in these organs, confirming the results obtained using PCR and ELISA, and indicating that the sample was contaminated with REV. These data clearly indicate that the screening of all commercial poultry vaccines for viruses is an important factor in assuring the biosafety of animal vaccines.

Development of a sensitive detection method for stressed E. coli O157:H7 in source and finished drinking water by culture-qPCR.

PubMed

Sen, Keya; L Sinclair, James; Boczek, Laura; Rice, Eugene W

2011-03-15

A sensitive and specific method that also demonstrates viability is of interest for detection of E. coli O157:H7 in drinking water. A combination of culture and qPCR was investigated. Two triplex qPCRs, one from a commercial source and another designed for this study were optimized from 5 different assays to be run on a single qPCR plate. The qPCR assays were specific for 33 E. coli O157:H7 strains tested and detected 500 cells spiked in a background of 10(8) nontarget bacterial cells. The qPCR detection was combined with an enrichment process using Presence Absence (P/A) broth to detect chlorine and starvation stressed cells. qPCR analysis performed post-enrichment allowed the detection of 3-4 cells/L as indicated by a sharp increase in fluorescence (lowering of Ct values) from pre-enrichment levels, demonstrating a 5-6 log increase in the number of cells. When six vulnerable untreated surface water samples were examined, only one was positive for viable E. coli O157:H7 cells. These results suggest that the culture-PCR procedure can be used for rapid detection of E. coli O157:H7 in drinking water.

Rapid diagnosis of common deletional α-thalassemia in the Chinese population by qPCR based on identical primer homologous fragments.

PubMed

Long, Ju

2016-05-01

In China, -(SEA), -α(3.7) and -α(4.2) are common deletional α-thalassemia alleles. Gap-PCR is the currently used detection method for these alleles, whose disadvantages include time-consuming procedure and increased potential for PCR product contamination. Therefore, this detection method needs to be improved. Based on identical-primer homologous fragments, a qPCR system was developed for deletional α-thalassemia genotyping, which was composed of a group of quantitatively-related primers and their corresponding probes plus two groups of qualitatively-related primers and their corresponding probes. In order to verify the accuracy of the qPCR system, known genotype samples and random samples are employed. The standard curve result demonstrated that designed primers and probes all yielded good amplification efficiency. In the tests of known genotype samples and random samples, sample detection results were consistent with verification results. In detecting αα, -(SEA), -α(3.7) and -α(4.2) alleles, deletional α-thalassemia alleles are accurately detected by this method. In addition, this method is provided with a wider detection range, greater speed and reduced PCR product contamination risk when compared with current common gap-PCR detection reagents. Copyright © 2016 Elsevier B.V. All rights reserved.

Comparison of detection methods for HPV status as a prognostic marker for loco-regional control after radiochemotherapy in patients with HNSCC.

PubMed

Linge, Annett; Schötz, Ulrike; Löck, Steffen; Lohaus, Fabian; von Neubeck, Cläre; Gudziol, Volker; Nowak, Alexander; Tinhofer, Inge; Budach, Volker; Sak, Ali; Stuschke, Martin; Balermpas, Panagiotis; Rödel, Claus; Bunea, Hatice; Grosu, Anca-Ligia; Abdollahi, Amir; Debus, Jürgen; Ganswindt, Ute; Lauber, Kirsten; Pigorsch, Steffi; Combs, Stephanie E; Mönnich, David; Zips, Daniel; Baretton, Gustavo B; Buchholz, Frank; Krause, Mechthild; Belka, Claus; Baumann, Michael

2018-04-01

To compare six HPV detection methods in pre-treatment FFPE tumour samples from patients with locally advanced head and neck squamous cell carcinoma (HNSCC) who received postoperative (N = 175) or primary (N = 90) radiochemotherapy. HPV analyses included detection of (i) HPV16 E6/E7 RNA, (ii) HPV16 DNA (PCR-based arrays, A-PCR), (iii) HPV DNA (GP5+/GP6+ qPCR, (GP-PCR)), (iv) p16 (immunohistochemistry, p16 IHC), (v) combining p16 IHC and the A-PCR result and (vi) combining p16 IHC and the GP-PCR result. Differences between HPV positive and negative subgroups were evaluated for the primary endpoint loco-regional control (LRC) using Cox regression. Correlation between the HPV detection methods was high (chi-squared test, p < 0.001). While p16 IHC analysis resulted in several false positive classifications, A-PCR, GP-PCR and the combination of p16 IHC and A-PCR or GP-PCR led to results comparable to RNA analysis. In both cohorts, Cox regression analyses revealed significantly prolonged LRC for patients with HPV positive tumours irrespective of the detection method. The most stringent classification was obtained by detection of HPV16 RNA, or combining p16 IHC with A-PCR or GP-PCR. This approach revealed the lowest rate of recurrence in patients with tumours classified as HPV positive and therefore appears most suited for patient stratification in HPV-based clinical studies. Copyright © 2017 Elsevier B.V. All rights reserved.

Evaluation of PCR Approaches for Detection of Bartonella bacilliformis in Blood Samples.

PubMed

Gomes, Cláudia; Martinez-Puchol, Sandra; Pons, Maria J; Bazán, Jorge; Tinco, Carmen; del Valle, Juana; Ruiz, Joaquim

2016-03-01

The lack of an effective diagnostic tool for Carrion's disease leads to misdiagnosis, wrong treatments and perpetuation of asymptomatic carriers living in endemic areas. Conventional PCR approaches have been reported as a diagnostic technique. However, the detection limit of these techniques is not clear as well as if its usefulness in low bacteriemia cases. The aim of this study was to evaluate the detection limit of 3 PCR approaches. We determined the detection limit of 3 different PCR approaches: Bartonella-specific 16S rRNA, fla and its genes. We also evaluated the viability of dry blood spots to be used as a sample transport system. Our results show that 16S rRNA PCR is the approach with a lowest detection limit, 5 CFU/μL, and thus, the best diagnostic PCR tool studied. Dry blood spots diminish the sensitivity of the assay. From the tested PCRs, the 16S rRNA PCR-approach is the best to be used in the direct blood detection of acute cases of Carrion's disease. However its use in samples from dry blood spots results in easier management of transport samples in rural areas, a slight decrease in the sensitivity was observed. The usefulness to detect by PCR the presence of low-bacteriemic or asymptomatic carriers is doubtful, showing the need to search for new more sensible techniques.

Quantitative detection method of Enterocytozoon hepatopenaei using TaqMan probe real-time PCR.

PubMed

Liu, Ya-Mei; Qiu, Liang; Sheng, An-Zhi; Wan, Xiao-Yuan; Cheng, Dong-Yuan; Huang, Jie

2018-01-01

A TaqMan probe and a pair of specific primers were selected from the small subunit ribosomal DNA (SSU rDNA) sequence of Enterocytozoon hepatopenaei (EHP); this real-time PCR assay was developed and optimized. It showed a good linearity in detecting standards of EHP SSU rDNA fragments from 4 × 10 2 to 4 × 10 8 copies/reaction using the established method. The detection limit of the qPCR method was as low as 4 × 10 1 copies per reaction, which was higher than the conventional PCR and SYBR Green I-based EHP qPCR reported. Using the qPCR assay, EHP was detected in four batches of slow-growing Penaeus vannamei specimens collected from Tianjin and Zhejiang Province in China was detected using qPCR. The results showed that all the hepatopancreas from the slow-growing P. vannamei specimens were detected as EHP-positive. EHP copies of hepatopancreas in some batches had a negative correlation with the body mass index (BMI) of shrimps; however, not all batches of specimens had this negative correlation between EHP copies of hepatopancreas and BMI. This qPCR technique is sensitive, specific and easy to perform (96 tests in <3 h), which provides technical support for the detection and prevention of EHP. Copyright © 2017 Elsevier Inc. All rights reserved.

Detection of pathogenic organisms in food, water, and body fluids

NASA Astrophysics Data System (ADS)

Wallace, William H.; Henley, Michael V.; Sayler, Gary S.

2002-06-01

The construction of specific bioluminescent bacteriophage for detection of pathogenic organism can be developed to overcome interferences in complex matrices such as food, water and body fluids. Detection and identification of bacteria often require several days and frequently weeks by standard methods of isolation, growth and biochemical test. Immunoassay detection often requires the expression of the bacterial toxin, which can lead to non-detection of cells that may express the toxin under conditions different from testing protocols. Immunoassays require production of a specific antibody to the agent for detection and interference by contaminants frequently affects results. PCR based detection may be inhibited by substances in complex matrices. Modified methods of the PCR technique, such as magnetic capture-hybridization PCR (MCH-PCR), appear to improve the technique by removing the DNA products away from the inhibitors. However, the techniques required for PCR-based detection are slow and the procedures require skilled personnel working with labile reagents. Our approach is based on transferring bioluminescence (lux) genes into a selected bacteriophage. Bacteriophages are bacterial viruses that are widespread in nature and often are genus and species specific. This specificity eliminates or reduces false positives in a bacteriophage assay. The phage recognizes a specific receptor molecule on the surface of a susceptible bacterium, attaches and then injects the viral nucleic acid into the cell. The injected viral genome is expressed and then replicated, generating numerous exact copies of the viral genetic material including the lux genes, often resulting in an increase in bioluminescence by several hundred fold.

Carbapenem-resistant Gram-negative bacteria associated with catheter-related bloodstream infections in three intensive care units in Egypt.

PubMed

Abdulall, Abeer K; Tawfick, Mahmoud M; El Manakhly, Arwa R; El Kholy, Amani

2018-06-23

We aimed to identify the carbapenem-resistant Gram-negative bacteria (GNB) causing catheter-related bloodstream infections (CRBSI) in intensive care units (ICU) in a tertiary care Egyptian hospital, to study their resistance mechanisms by phenotypic and genetic tests, and to use ERIC-PCR for assessing their relatedness. The study was conducted over 2 years in three ICUs in a tertiary care hospital in Egypt during 2015-2016. We identified 194 bloodstream infections (BSIs); 130 (67.01%) were caused by GNB, of which 57 were isolated from CRBSI patients (73.84%). Identification of isolates was performed using conventional methods and MALDI-TOF MS. Antimicrobial susceptibility testing (AST) was done by disc diffusion following CLSI guidelines. Phenotypic detection of carbapenemases enzymes activity was by modified Hodge test and the Carba-NP method. Isolates were investigated for the most common carbapenemases encoding genes bla KPC , bla NDM , and bla OXA-48 using multiplex PCR. Molecular typing of carbapenem-resistant isolates was done by ERIC-PCR followed by sequencing of common resistance genes. The overall rate of CRBSI in our study was 3.6 per 1000 central venous catheter (CVC) days. Among 57 Gram-negative CRBSI isolates, Klebsiella pneumoniae (K. pneumoniae) was the most frequently isolated (27/57; 47.4%), of which more than 70% were resistant to Meropenem. Phenotypic tests for carbapenemases showed that 37.9% of isolates were positive by modified Hodge test and 63.8% by Carba-NP detection. Multiplex PCR assay detected the bla NDM in 28.6% of the isolates and bla KPC in 26.8%, bla NDM and bla KPC were detected together in the same isolate in 5.6%, while bla OXA-48 -like were not detected. ERIC-PCR detected limited genetic relatedness between K. pneumoniae isolates. Elevated resistance rates were observed to all antibiotics including carbapenems among K. pneumoniae isolates causing CRBSI. ERIC-PCR showed that the resistant isolates were mainly polyclonal. Our results call for reinforcement of antimicrobial stewardship and measures to prevent CRBSI.

Specific detection of rinderpest virus by real-time reverse transcription-PCR in preclincal and clinical samples of experimentally infected cattle

USDA-ARS?s Scientific Manuscript database

A highly sensitive detection test for Rinderpest virus (RPV), based on a real-time reverse transcription-PCR (RT-PR) system, was developed. Five different RPV genomic targets were examined, and one was selected and optimized to detect viral RNA in infected tissue culture fluid with a level of detec...

RT-PCR for detection of all seven genotypes of Lyssavirus genus.

PubMed

Vázquez-Morón, S; Avellón, A; Echevarría, J E

2006-08-01

The Lyssavirus genus includes seven species or genotypes named 1-7. Rabies genotypes correlate with geographical distribution and specific hosts. Co-circulation of different lyssaviruses, imported cases, and the presence of unknown viruses, such as Aravan, Khujand, Irkut and West Caucasian Bat Virus, make it necessary to use generic methods able to detect all lyssaviruses. Primer sequences were chosen from conserved regions in all genotypes in order to optimise a generic RT-PCR. Serial dilutions of 12 RNA extracts from all seven Lyssavirus genotypes were examined to compare the sensitivity of the RT-PCR standardised in this study with a published RT-PCR optimised for EBLV1 detection and capable of amplifying RNA from all seven lyssaviruses. All seven genotypes were detected by both RT-PCRs, however, the sensitivity was higher with the new version of the test. Twenty samples submitted for rabies diagnosis were tested by the new RT-PCR. Eight out of 20 samples from six dogs, one horse and one bat were found positive, in agreement with immunofluorescence results. Seven samples from terrestrial mammals were genotype 1 and one from a bat was genotype 5. In conclusion, this method can be used to complement immunofluorescence for the diagnosis of rabies, enabling the detection of unexpected lyssaviruses during rabies surveillance.

International Study to Evaluate PCR Methods for Detection of Trypanosoma cruzi DNA in Blood Samples from Chagas Disease Patients

PubMed Central

Schijman, Alejandro G.; Bisio, Margarita; Orellana, Liliana; Sued, Mariela; Duffy, Tomás; Mejia Jaramillo, Ana M.; Cura, Carolina; Auter, Frederic; Veron, Vincent; Qvarnstrom, Yvonne; Deborggraeve, Stijn; Hijar, Gisely; Zulantay, Inés; Lucero, Raúl Horacio; Velazquez, Elsa; Tellez, Tatiana; Sanchez Leon, Zunilda; Galvão, Lucia; Nolder, Debbie; Monje Rumi, María; Levi, José E.; Ramirez, Juan D.; Zorrilla, Pilar; Flores, María; Jercic, Maria I.; Crisante, Gladys; Añez, Néstor; De Castro, Ana M.; Gonzalez, Clara I.; Acosta Viana, Karla; Yachelini, Pedro; Torrico, Faustino; Robello, Carlos; Diosque, Patricio; Triana Chavez, Omar; Aznar, Christine; Russomando, Graciela; Büscher, Philippe; Assal, Azzedine; Guhl, Felipe; Sosa Estani, Sergio; DaSilva, Alexandre; Britto, Constança; Luquetti, Alejandro; Ladzins, Janis

2011-01-01

Background A century after its discovery, Chagas disease still represents a major neglected tropical threat. Accurate diagnostics tools as well as surrogate markers of parasitological response to treatment are research priorities in the field. The purpose of this study was to evaluate the performance of PCR methods in detection of Trypanosoma cruzi DNA by an external quality evaluation. Methodology/Findings An international collaborative study was launched by expert PCR laboratories from 16 countries. Currently used strategies were challenged against serial dilutions of purified DNA from stocks representing T. cruzi discrete typing units (DTU) I, IV and VI (set A), human blood spiked with parasite cells (set B) and Guanidine Hidrochloride-EDTA blood samples from 32 seropositive and 10 seronegative patients from Southern Cone countries (set C). Forty eight PCR tests were reported for set A and 44 for sets B and C; 28 targeted minicircle DNA (kDNA), 13 satellite DNA (Sat-DNA) and the remainder low copy number sequences. In set A, commercial master mixes and Sat-DNA Real Time PCR showed better specificity, but kDNA-PCR was more sensitive to detect DTU I DNA. In set B, commercial DNA extraction kits presented better specificity than solvent extraction protocols. Sat-DNA PCR tests had higher specificity, with sensitivities of 0.05–0.5 parasites/mL whereas specific kDNA tests detected 5.10−3 par/mL. Sixteen specific and coherent methods had a Good Performance in both sets A and B (10 fg/µl of DNA from all stocks, 5 par/mL spiked blood). The median values of sensitivities, specificities and accuracies obtained in testing the Set C samples with the 16 tests determined to be good performing by analyzing Sets A and B samples varied considerably. Out of them, four methods depicted the best performing parameters in all three sets of samples, detecting at least 10 fg/µl for each DNA stock, 0.5 par/mL and a sensitivity between 83.3–94.4%, specificity of 85–95%, accuracy of 86.8–89.5% and kappa index of 0.7–0.8 compared to consensus PCR reports of the 16 good performing tests and 63–69%, 100%, 71.4–76.2% and 0.4–0.5, respectively compared to serodiagnosis. Method LbD2 used solvent extraction followed by Sybr-Green based Real time PCR targeted to Sat-DNA; method LbD3 used solvent DNA extraction followed by conventional PCR targeted to Sat-DNA. The third method (LbF1) used glass fiber column based DNA extraction followed by TaqMan Real Time PCR targeted to Sat-DNA (cruzi 1/cruzi 2 and cruzi 3 TaqMan probe) and the fourth method (LbQ) used solvent DNA extraction followed by conventional hot-start PCR targeted to kDNA (primer pairs 121/122). These four methods were further evaluated at the coordinating laboratory in a subset of human blood samples, confirming the performance obtained by the participating laboratories. Conclusion/Significance This study represents a first crucial step towards international validation of PCR procedures for detection of T. cruzi in human blood samples. PMID:21264349

High Throughput Multiplex PCR and Probe-based Detection with Luminex Beads for Seven Intestinal Parasites

PubMed Central

Taniuchi, Mami; Verweij, Jaco J.; Noor, Zannatun; Sobuz, Shihab U.; van Lieshout, Lisette; Petri, William A.; Haque, Rashidul; Houpt, Eric R.

2011-01-01

Polymerase chain reaction (PCR) assays for intestinal parasites are increasingly being used on fecal DNA samples for enhanced specificity and sensitivity of detection. Comparison of these tests against microscopy and copro-antigen detection has been favorable, and substitution of PCR-based assays for the ova and parasite stool examination is a foreseeable goal for the near future. One challenge is the diverse list of protozoan and helminth parasites. Several existing real-time PCR assays for the major intestinal parasites—Cryptosporidium spp., Giardia intestinalis, Entamoeba histolytica, Ancylostoma duodenale, Ascaris lumbricoides, Necator americanus, and Strongyloides stercoralis—were adapted into a high throughput protocol. The assay involves two multiplex PCR reactions, one with specific primers for the protozoa and one with specific primers for the helminths, after which PCR products are hybridized to beads linked to internal oligonucleotide probes and detected on a Luminex platform. When compared with the parent multiplex real-time PCR assays, this multiplex PCR-bead assay afforded between 83% and 100% sensitivity and specificity on a total of 319 clinical specimens. In conclusion, this multiplex PCR-bead protocol provides a sensitive diagnostic screen for a large panel of intestinal parasites. PMID:21292910

PCR Inhibition of a Quantitative PCR for Detection of Mycobacterium avium Subspecies Paratuberculosis DNA in Feces: Diagnostic Implications and Potential Solutions

PubMed Central

Acharya, Kamal R.; Dhand, Navneet K.; Whittington, Richard J.; Plain, Karren M.

2017-01-01

Molecular tests such as polymerase chain reaction (PCR) are increasingly being applied for the diagnosis of Johne’s disease, a chronic intestinal infection of ruminants caused by Mycobacterium avium subspecies paratuberculosis (MAP). Feces, as the primary test sample, presents challenges in terms of effective DNA isolation, with potential for PCR inhibition and ultimately for reduced analytical and diagnostic sensitivity. However, limited evidence is available regarding the magnitude and diagnostic implications of PCR inhibition for the detection of MAP in feces. This study aimed to investigate the presence and diagnostic implications of PCR inhibition in a quantitative PCR assay for MAP (High-throughput Johne’s test) to investigate the characteristics of samples prone to inhibition and to identify measures that can be taken to overcome this. In a study of fecal samples derived from a high prevalence, endemically infected cattle herd, 19.94% of fecal DNA extracts showed some evidence of inhibition. Relief of inhibition by a five-fold dilution of the DNA extract led to an average increase in quantification of DNA by 3.3-fold that consequently increased test sensitivity of the qPCR from 55 to 80% compared to fecal culture. DNA extracts with higher DNA and protein content had 19.33 and 10.94 times higher odds of showing inhibition, respectively. The results suggest that the current test protocol is sensitive for herd level diagnosis of Johne’s disease but that test sensitivity and individual level diagnosis could be enhanced by relief of PCR inhibition, achieved by five-fold dilution of the DNA extract. Furthermore, qualitative and quantitative parameters derived from absorbance measures of DNA extracts could be useful for prediction of inhibitory fecal samples. PMID:28210245

Analytical Performance of Four Polymerase Chain Reaction (PCR) and Real Time PCR (qPCR) Assays for the Detection of Six Leishmania Species DNA in Colombia

PubMed Central

León, Cielo M.; Muñoz, Marina; Hernández, Carolina; Ayala, Martha S.; Flórez, Carolina; Teherán, Aníbal; Cubides, Juan R.; Ramírez, Juan D.

2017-01-01

Leishmaniasis comprises a spectrum of parasitic diseases caused by protozoans of the genus Leishmania. Molecular tools have been widely employed for the detection of Leishmania due to its high sensitivity and specificity. However, the analytical performance of molecular platforms as PCR and real time PCR (qPCR) including a wide variety of molecular markers has never been evaluated. Herein, the aim was to evaluate the analytical performance of 4 PCR-based assays (designed on four different targets) and applied on conventional and real-time PCR platforms. We evaluated the analytical performance of conventional PCR and real time PCR, determining exclusivity and inclusivity, Anticipated Reportable Range (ARR), limit of detection (LoD) and accuracy using primers directed to kDNA, HSP70, 18S and ITS-1 targets. We observed that the kDNA was the most sensitive but does not meet the criterion of exclusivity. The HSP70 presented a higher LoD in conventional PCR and qPCR in comparison with the other markers (1 × 101 and 1 × 10-1 equivalent parasites/mL respectively) and had a higher coefficient of variation in qPCR. No statistically significant differences were found between the days of the test with the four molecular markers. The present study revealed that the 18S marker presented the best performance in terms of analytical sensitivity and specificity for the qPCR in the species tested (species circulating in Colombia). Therefore, we recommend to explore the analytical and diagnostic performance in future studies using a broader number of species across America. PMID:29046670

Analytical Performance of Four Polymerase Chain Reaction (PCR) and Real Time PCR (qPCR) Assays for the Detection of Six Leishmania Species DNA in Colombia.

PubMed

León, Cielo M; Muñoz, Marina; Hernández, Carolina; Ayala, Martha S; Flórez, Carolina; Teherán, Aníbal; Cubides, Juan R; Ramírez, Juan D

2017-01-01

Leishmaniasis comprises a spectrum of parasitic diseases caused by protozoans of the genus Leishmania . Molecular tools have been widely employed for the detection of Leishmania due to its high sensitivity and specificity. However, the analytical performance of molecular platforms as PCR and real time PCR (qPCR) including a wide variety of molecular markers has never been evaluated. Herein, the aim was to evaluate the analytical performance of 4 PCR-based assays (designed on four different targets) and applied on conventional and real-time PCR platforms. We evaluated the analytical performance of conventional PCR and real time PCR, determining exclusivity and inclusivity, Anticipated Reportable Range (ARR), limit of detection (LoD) and accuracy using primers directed to kDNA, HSP70, 18S and ITS-1 targets. We observed that the kDNA was the most sensitive but does not meet the criterion of exclusivity. The HSP70 presented a higher LoD in conventional PCR and qPCR in comparison with the other markers (1 × 10 1 and 1 × 10 -1 equivalent parasites/mL respectively) and had a higher coefficient of variation in qPCR. No statistically significant differences were found between the days of the test with the four molecular markers. The present study revealed that the 18S marker presented the best performance in terms of analytical sensitivity and specificity for the qPCR in the species tested (species circulating in Colombia). Therefore, we recommend to explore the analytical and diagnostic performance in future studies using a broader number of species across America.

Evaluation of an Improved U.S. Food and Drug Administration Method for the Detection of Cyclospora cayetanensis in Produce Using Real-Time PCR.

PubMed

Murphy, Helen R; Lee, Seulgi; da Silva, Alexandre J

2017-07-01

Cyclospora cayetanensis is a protozoan parasite that causes human diarrheal disease associated with the consumption of fresh produce or water contaminated with C. cayetanensis oocysts. In the United States, foodborne outbreaks of cyclosporiasis have been linked to various types of imported fresh produce, including cilantro and raspberries. An improved method was developed for identification of C. cayetanensis in produce at the U.S. Food and Drug Administration. The method relies on a 0.1% Alconox produce wash solution for efficient recovery of oocysts, a commercial kit for DNA template preparation, and an optimized TaqMan real-time PCR assay with an internal amplification control for molecular detection of the parasite. A single laboratory validation study was performed to assess the method's performance and compare the optimized TaqMan real-time PCR assay and a reference nested PCR assay by examining 128 samples. The samples consisted of 25 g of cilantro or 50 g of raspberries seeded with 0, 5, 10, or 200 C. cayetanensis oocysts. Detection rates for cilantro seeded with 5 and 10 oocysts were 50.0 and 87.5%, respectively, with the real-time PCR assay and 43.7 and 94.8%, respectively, with the nested PCR assay. Detection rates for raspberries seeded with 5 and 10 oocysts were 25.0 and 75.0%, respectively, with the real-time PCR assay and 18.8 and 68.8%, respectively, with the nested PCR assay. All unseeded samples were negative, and all samples seeded with 200 oocysts were positive. Detection rates using the two PCR methods were statistically similar, but the real-time PCR assay is less laborious and less prone to amplicon contamination and allows monitoring of amplification and analysis of results, making it more attractive to diagnostic testing laboratories. The improved sample preparation steps and the TaqMan real-time PCR assay provide a robust, streamlined, and rapid analytical procedure for surveillance, outbreak response, and regulatory testing of foods for detection of C. cayetanensis.

A multiplex PCR method for the simultaneous detection of three viruses associated with canine viral enteric infections.

PubMed

Deng, Xiaoyu; Zhang, Jiali; Su, Jiazi; Liu, Hao; Cong, Yanlong; Zhang, Lei; Zhang, Kemeng; Shi, Ning; Lu, Rongguang; Yan, Xijun

2018-04-19

The aim of this study was to establish a multiplex PCR (mPCR) method that can simultaneously detect canine parvovirus (CPV-2), canine coronavirus (CCoV) and canine adenovirus (CAV), thereby eliminating the need to detect these pathogens individually. Based on conserved regions in the genomes of these three viruses, the VP2 gene of CPV-2, the endoribonuclease nsp15 gene of CCoV, and the 52K gene of CAV were selected for primer design. The specificity of the mPCR results showed no amplification of canine distemper virus (CDV), canine parainfluenza virus (CPIV), or pseudorabies virus (PRV), indicating that the method had good specificity. A sensitivity test showed that the detection limit of the mPCR method was 1 × 10 4 viral copies. A total of 63 rectal swabs from dogs with diarrheal symptoms were evaluated using mPCR and routine PCR. The ratio of positive samples to total samples for CPV-2, CCoV, and CAV was 55.6% (35/63) for mPCR and 55.6% (35/63) for routine PCR. Thirty-five positive samples were detected by both methods, for a coincidence ratio of 100%. This mPCR method can simultaneously detect CCoV (CCoV-II), CAV (CAV-1, CAV-2) and CPV-2 (CPV-2a, CPV-2b, CPV-2c), which are associated with viral enteritis, thereby providing an efficient, inexpensive, specific, and accurate new tool for clinical diagnosis and laboratory epidemiological investigations.

Analytical validation of a reverse transcriptase droplet digital PCR (RT-ddPCR) for quantitative detection of infectious hematopoietic necrosis virus

USGS Publications Warehouse

Jia, Peng; Purcell, Maureen; Pan, Guang; Wang, Jinjin; Kan, Shifu; Liu, Yin; Zheng, Xiaocong; SHi, Xiujie; He, Junqiang; Yu, Li; Hua, Qunyi; Lu, Tikang; Lan, Wensheng; Winton, James; Jin, Ningyi; Liu, Hong

2017-01-01

Infectious hematopoietic necrosis virus (IHNV) is an important pathogen of salmonid fishes. A validated universal reverse transcriptase quantitative PCR (RT-qPCR) assay that can quantify levels of IHNV in fish tissues has been previously reported. In the present study, we adapted the published set of IHNV primers and probe for use in a reverse-transcriptase droplet digital PCR (RT-ddPCR) assay for quantification of the virus in fish tissue samples. The RT-ddPCR and RT-qPCR assays detected 13 phylogenetically diverse IHNV strains, but neither assay produced detectable amplification when RNA from other fish viruses was used. The RT-ddPCR assay had a limit of detection (LOD) equating to 2.2 plaque forming units (PFU)/μl while the LOD for the RT-qPCR was 0.2 PFU/μl. Good agreement (69.4–100%) between assays was observed when used to detect IHNV RNA in cell culture supernatant and tissues from IHNV infected rainbow trout (Oncorhynchus mykiss) and arctic char (Salvelinus alpinus). Estimates of RNA copy number produced by the two assays were significantly correlated but the RT-qPCR consistently produced higher estimates than the RT-ddPCR. The analytical properties of the N gene RT-ddPCR test indicated that this method may be useful to assess IHNV RNA copy number for research and diagnostic purposes. Future work is needed to establish the within and between laboratory diagnostic performance of the RT-ddPCR assay.

Diagnosis of Barmah Forest Virus Infection by a Nested Real-Time SYBR Green RT-PCR Assay

PubMed Central

Hueston, Linda; Toi, Cheryl S.; Jeoffreys, Neisha; Sorrell, Tania; Gilbert, Gwendolyn

2013-01-01

Barmah Forest virus (BFV) is a mosquito borne (+) ssRNA alphavirus found only in Australia. It causes rash, myalgia and arthralgia in humans and is usually diagnosed serologically. We developed a real-time PCR assay to detect BFV in an effort to improve diagnosis early in the course of infection. The limit of detection was 16 genome equivalents with a specificity of 100%. Fifty five serum samples from BFV-infected patients were tested by the PCR. 52 of 53 antibody-positive samples were PCR negative. Two culture-positive (neutralizing antibody negative) samples were positive on first round PCR, while one sample (IgM and neutralizing antibody strongly positive, IgG negative) was positive on second round PCR, suggesting that viral RNA is detectable and transiently present in early infection. PCR can provide results faster than culture, is capable of high throughput and by sequencing the PCR product strain variants can be characterized. PMID:23935816

A multiplex PCR method for the identification of commercially important salmon and trout species (Oncorhynchus and Salmo) in North America.

PubMed

Rasmussen Hellberg, Rosalee S; Morrissey, Michael T; Hanner, Robert H

2010-09-01

The purpose of this study was to develop a species-specific multiplex polymerase chain reaction (PCR) method that allows for the detection of salmon species substitution on the commercial market. Species-specific primers and TaqMan® probes were developed based on a comprehensive collection of mitochondrial 5' cytochrome c oxidase subunit I (COI) deoxyribonucleic acid (DNA) "barcode" sequences. Primers and probes were combined into multiplex assays and tested for specificity against 112 reference samples representing 25 species. Sensitivity and linearity tests were conducted using 10-fold serial dilutions of target DNA (single-species samples) and DNA admixtures containing the target species at levels of 10%, 1.0%, and 0.1% mixed with a secondary species. The specificity tests showed positive signals for the target DNA in both real-time and conventional PCR systems. Nonspecific amplification in both systems was minimal; however, false positives were detected at low levels (1.2% to 8.3%) in conventional PCR. Detection levels were similar for admixtures and single-species samples based on a 30 PCR cycle cut-off, with limits of 0.25 to 2.5 ng (1% to 10%) in conventional PCR and 0.05 to 5.0 ng (0.1% to 10%) in real-time PCR. A small-scale test with food samples showed promising results, with species identification possible even in heavily processed food items. Overall, this study presents a rapid, specific, and sensitive method for salmon species identification that can be applied to mixed-species and heavily processed samples in either conventional or real-time PCR formats. This study provides a newly developed method for salmon and trout species identification that will assist both industry and regulatory agencies in the detection and prevention of species substitution. This multiplex PCR method allows for rapid, high-throughput species identification even in heavily processed and mixed-species samples. An inter-laboratory study is currently being carried out to assess the ability of this method to identify species in a variety of commercial salmon and trout products.

Competitive allele-specific TaqMan PCR (Cast-PCR) is a sensitive, specific and fast method for BRAF V600 mutation detection in Melanoma patients

PubMed Central

Barbano, Raffaela; Pasculli, Barbara; Coco, Michelina; Fontana, Andrea; Copetti, Massimiliano; Rendina, Michelina; Valori, Vanna Maria; Graziano, Paolo; Maiello, Evaristo; Fazio, Vito Michele; Parrella, Paola

2015-01-01

BRAF codon 600 mutation testing of melanoma patients is mandatory for the choice of the most appropriate therapy in the clinical setting. Competitive allele specific TaqMan PCR (Cast-PCR) technology allows not only the selective amplification of minor alleles, but it also blocks the amplification of non-mutant allele. We genotyped codon 600 of the BRAF gene in 54 patients’ samples by Cast-PCR and bidirectional direct sequence analysis. All the mutations detected by sequencing were also identified by Cast-PCR. In addition, Cast-PCR assay detected four samples carrying mutations and was able to clearly identify two mutations of uncertain interpretation by Sanger sequencing. The limit of detection of Cast-PCR was evaluated by constructing dilution curves of BRAFV600E and BRAFV600K mutated clinical samples mixed with a not-mutated specimens. Both mutations could be detected until a 1:100 mutated/not mutated ratio. Cloning and sequencing of the clones was used to confirm mutations on representative discrepant cases. Cast PCR performances were not affected by intratumour heterogeneity, and less affected by melanin content. Our results indicate that Cast-PCR is a reliable diagnostic tool for the identification of melanoma patients as eligible to be treated with TKIs and might be implemented in the clinical setting as elective screening method. PMID:26690267

Rapid detection of Porcine circovirus 2 by recombinase polymerase amplification.

PubMed

Wang, Jianchang; Wang, Jinfeng; Liu, Libing; Li, Ruiwen; Yuan, Wanzhe

2016-09-01

Porcine circovirus-associated disease, caused primarily by Porcine circovirus 2 (PCV-2), has become endemic in many pig-producing countries and has resulted in significant economic losses to the swine industry worldwide. Tests for PCV-2 infection include PCR, nested PCR, competitive PCR, and real-time PCR (rtPCR). Recombinase polymerase amplification (RPA) has emerged as an isothermal gene amplification technology for the molecular detection of infectious disease agents. RPA is performed at a constant temperature and therefore can be carried out in a water bath. In addition, RPA is completed in ~30 min, much faster than PCR, which usually takes >60 min. We developed a RPA-based method for the detection of PCV-2. The detection limit of RPA was 10(2) copies of PCV-2 genomic DNA. RPA showed the same sensitivity as rtPCR but was 10 times more sensitive than conventional PCR. Successful amplification of PCV-2 DNA, but not other viral templates, demonstrated high specificity of the RPA assay. This method was also validated using clinical samples. The results showed that the RPA assay had a diagnostic agreement rate of 93.7% with conventional PCR and 100% with rtPCR. These findings suggest that the RPA assay is a simple, rapid, and cost-effective method for PCV-2 detection, which could be potentially applied in clinical diagnosis and field surveillance of PCV-2 infection. © 2016 The Author(s).

Development of a reverse transcription polymerase chain reaction method for yellow fever virus detection.

PubMed

Méndez, María C; Domingo, Cristina; Tenorio, Antonio; Pardo, Lissethe C; Rey, Gloria J; Méndez, Jairo A

2013-09-01

Yellow fever is considered a re-emerging disease and is endemic in tropical regions of Africa and South America. At present, there are no standardized or commercialized kits available for yellow fever virus detection. Therefore, diagnosis must be made by time-consuming routine techniques, and sometimes, the virus or its proteins are not detected. Furthermore, co-circulation with other flaviviruses, including dengue virus, increases the difficulty of diagnosis. To develop a specific reverse transcriptase-polymerase chain reaction (RT-PCR) and nested PCR-based assay to improve the detection and diagnosis of yellow fever virus using both serum and fresh tissue samples. RT-PCR primers were designed to amplify a short fragment of all yellow fever virus genotypes reported. A second set of primers was used in a nested PCR to increase sensitivity. Thirty-three clinical samples were tested with the standardized reaction. The expected amplicon was obtained in 25 out of 33 samples analyzed using this approach, and 2 more samples tested positive after a subsequent nested PCR approach. This improved technique not only ensures the specific detection of a wide range of yellow fever virus genotypes but also may increase the sensitivity of detection by introducing a second round of amplification, allowing a rapid differential diagnosis between dengue and yellow fever infection, which is required for effective surveillance and opportune epidemiologic measures.

Molecular diagnosis of lyssaviruses and sequence comparison of Australian bat lyssavirus samples.

PubMed

Foord, A J; Heine, H G; Pritchard, L I; Lunt, R A; Newberry, K M; Rootes, C L; Boyle, D B

2006-07-01

To evaluate and implement molecular diagnostic tests for the detection of lyssaviruses in Australia. A published hemi-nested reverse transcriptase polymerase chain reaction (RT-PCR) for the detection of all lyssavirus genotypes was modified to a fully nested RT-PCR format and compared with the original assay. TaqMan assays for the detection of Australian bat lyssavirus (ABLV) were compared with both the nested and hemi-nested RT-PCR assays. The sequences of RT-PCR products were determined to assess sequence variations of the target region (nucleocapsid gene) in samples of ABLV originating from different regions. The nested RT-PCR assay was highly analytically specific, and at least as analytically sensitive as the hemi-nested assay. The TaqMan assays were highly analytically specific and more analytically sensitive than either RT-PCR assay, with a detection level of approximately 10 genome equivalents per microl. Sequence of the first 544 nucleotides of the nucleocapsid protein coding sequence was obtained from all samples of ABLV received at Australian Animal Health Laboratory during the study period. The nested RT-PCR provided a means for molecular diagnosis of all tested genotypes of lyssavirus including classical rabies virus and Australian bat lyssavirus. The published TaqMan assay proved to be superior to the RT-PCR assays for the detection of ABLV in terms of analytical sensitivity. The TaqMan assay would also be faster and cross contamination is less likely. Nucleotide sequence analyses of samples of ABLV from a wide geographical range in Australia demonstrated the conserved nature of this region of the genome and therefore the suitability of this region for molecular diagnosis.

Development of reverse transcription-PCR assays specific for detection of equine encephalitis viruses.

PubMed

Linssen, B; Kinney, R M; Aguilar, P; Russell, K L; Watts, D M; Kaaden, O R; Pfeffer, M

2000-04-01

Specific and sensitive reverse transcription-PCR (RT-PCR) assays were developed for the detection of eastern, western, and Venezuelan equine encephalitis viruses (EEE, WEE, and VEE, respectively). Tests for specificity included all known alphavirus species. The EEE-specific RT-PCR amplified a 464-bp region of the E2 gene exclusively from 10 different EEE strains from South and North America with a sensitivity of about 3,000 RNA molecules. In a subsequent nested PCR, the specificity was confirmed by the amplification of a 262-bp fragment, increasing the sensitivity of this assay to approximately 30 RNA molecules. The RT-PCR for WEE amplified a fragment of 354 bp from as few as 2,000 RNA molecules. Babanki virus, as well as Mucambo and Pixuna viruses (VEE subtypes IIIA and IV), were also amplified. However, the latter viruses showed slightly smaller fragments of about 290 and 310 bp, respectively. A subsequent seminested PCR amplified a 195-bp fragment only from the 10 tested strains of WEE from North and South America, rendering this assay virus specific and increasing its sensitivity to approximately 20 RNA molecules. Because the 12 VEE subtypes showed too much divergence in their 26S RNA nucleotide sequences to detect all of them by the use of nondegenerate primers, this assay was confined to the medically important and closely related VEE subtypes IAB, IC, ID, IE, and II. The RT-PCR-seminested PCR combination specifically amplified 342- and 194-bp fragments of the region covering the 6K gene in VEE. The sensitivity was 20 RNA molecules for subtype IAB virus and 70 RNA molecules for subtype IE virus. In addition to the subtypes mentioned above, three of the enzootic VEE (subtypes IIIB, IIIC, and IV) showed the specific amplicon in the seminested PCR. The practicability of the latter assay was tested with human sera gathered as part of the febrile illness surveillance in the Amazon River Basin of Peru near the city of Iquitos. All of the nine tested VEE-positive sera showed the expected 194-bp amplicon of the VEE-specific RT-PCR-seminested PCR.

Virological Diagnosis of Central Nervous System Infections by Use of PCR Coupled with Mass Spectrometry Analysis of Cerebrospinal Fluid Samples

PubMed Central

Lévêque, Nicolas; Legoff, Jérôme; Mengelle, Catherine; Mercier-Delarue, Séverine; N'guyen, Yohan; Renois, Fanny; Tissier, Fabien; Simon, François; Izopet, Jacques

2014-01-01

Viruses are the leading cause of central nervous system (CNS) infections, ahead of bacteria, parasites, and fungal agents. A rapid and comprehensive virologic diagnostic testing method is needed to improve the therapeutic management of hospitalized pediatric or adult patients. In this study, we assessed the clinical performance of PCR amplification coupled with electrospray ionization-time of flight mass spectrometry analysis (PCR-MS) for the diagnosis of viral CNS infections. Three hundred twenty-seven cerebrospinal fluid (CSF) samples prospectively tested by routine PCR assays between 2004 and 2012 in two university hospital centers (Toulouse and Reims, France) were retrospectively analyzed by PCR-MS analysis using primers targeted to adenovirus, human herpesviruses 1 to 8 (HHV-1 to -8), polyomaviruses BK and JC, parvovirus B19, and enteroviruses (EV). PCR-MS detected single or multiple virus infections in 190 (83%) of the 229 samples that tested positive by routine PCR analysis and in 10 (10.2%) of the 98 samples that tested negative. The PCR-MS results correlated well with herpes simplex virus 1 (HSV-1), varicella-zoster virus (VZV), and EV detection by routine PCR assays (kappa values [95% confidence intervals], 0.80 [0.69 to 0.92], 0.85 [0.71 to 0.98], and 0.84 [0.78 to 0.90], respectively), whereas a weak correlation was observed with Epstein-Barr virus (EBV) (0.34 [0.10 to 0.58]). Twenty-six coinfections and 16 instances of uncommon neurotropic viruses (HHV-7 [n = 13], parvovirus B19 [n = 2], and adenovirus [n = 1]) were identified by the PCR-MS analysis, whereas only 4 coinfections had been prospectively evidenced using routine PCR assays (P < 0.01). In conclusion, our results demonstrated that PCR-MS analysis is a valuable tool to identify common neurotropic viruses in CSF (with, however, limitations that were identified regarding EBV and EV detection) and may be of major interest in better understanding the clinical impact of multiple or neglected viral neurological infections. PMID:24197874

Use of a real time PCR assay for detection of the ctxA gene of Vibrio cholerae in an environmental survey of Mobile Bay.

PubMed

Blackstone, George M; Nordstrom, Jessica L; Bowen, Michael D; Meyer, Richard F; Imbro, Paula; DePaola, Angelo

2007-02-01

Toxigenic Vibrio cholerae, the etiological agent of cholera, is a natural inhabitant of the marine environment and causes severe diarrheal disease affecting thousands of people each year in developing countries. It is the subject of extensive testing of shrimp produced and exported from these countries. We report the development of a real time PCR (qPCR) assay to detect the gene encoding cholera toxin, ctxA, found in toxigenic V. cholerae strains. This assay was tested against DNA isolated from soil samples collected from diverse locations in the US, a panel of eukaryotic DNA from various sources, and prokaryotic DNA from closely related and unrelated bacterial sources. Only Vibrio strains known to contain ctxA generated a fluorescent signal with the 5' nuclease probe targeting the ctxA gene, thus confirming the specificity of the assay. In addition, the assay was quantitative in pure culture across a six-log dynamic range down to <10 CFU per reaction. To test the robustness of this assay, oysters, aquatic sediments, and seawaters from Mobile Bay, AL, were analyzed by qPCR and traditional culture methods. The assay was applied to overnight alkaline peptone water enrichments of these matrices after boiling the enrichments for 10 min. Toxigenic V. cholerae strains were not detected by either qPCR or conventional methods in the 16 environmental samples examined. A novel exogenous internal amplification control developed by us to prevent false negatives identified the samples that were inhibitory to the PCR. This assay, with the incorporated internal control, provides a highly specific, sensitive, and rapid detection method for the detection of toxigenic strains of V. cholerae.

Comparison of nonstructural protein-1 antigen detection by rapid and enzyme-linked immunosorbent assay test and its correlation with polymerase chain reaction for early diagnosis of dengue

PubMed Central

Gaikwad, Seema; Sawant, Sandhya S.; Shastri, Jayanthi S.

2017-01-01

INTRODUCTION: Early diagnosis of dengue is important for appropriate clinical management and vector control. Different serological tests based on the principle of immunochromatography and enzyme-linked immunosorbent assay (ELISA) are commonly used for detection of antigen and antibodies of dengue virus. The performance of these tests depends on the sensitivity and specificity. Hence, the study was undertaken to compare nonstructural protein-1 (NS1) antigen detection by rapid and ELISA with real-time polymerase chain reaction (RT-PCR) for diagnosis of dengue. MATERIALS AND METHODS: Prospective laboratory study was carried out on sera samples (n = 200) from clinically suspected cases of dengue. The sera samples were subjected for NS1 antigen detection test by rapid test, NS1 ELISA, and RT-PCR. The results of rapid and ELISA tests were compared with real Time PCR. RESULTS: The sensitivity, specificity, positive, and negative predictive value of rapid dengue NS1 antigen test were 81.5%, 66.7%, 78.2%, and 71.1%, respectively whereas that of NS1 ELISA were 89.9%, 100%, 100%, and 94%, respectively. Concordance of Rapid NS1 and NS1 ELISA with PCR was 75.5% and 94%. DISCUSSION AND CONCLUSION: NS1 antigen ELISA can be implemented in diagnostic laboratories for diagnosis of dengue in the acute phase of illness. The test also has great potential value for use in epidemic situations, as it could facilitate the early screening of patients and limit disease expansion. PMID:28706387

Comparative evaluation of molecular diagnostic tests for Nucleospora salmonis and prevalence in migrating juvenile salmonids from the Snake River, USA

USGS Publications Warehouse

Badil, Samantha; Elliott, Diane G.; Kurobe, Tomofumi; Hedrick, Ronald P.; Clemens, Kathy; Blair, Marilyn; Purcell, Maureen K.

2011-01-01

Nucleospora salmonis is an intranuclear microsporidian that primarily infects lymphoblast cells and contributes to chronic lymphoblastosis and a leukemia-like condition in a range of salmonid species. The primary goal of this study was to evaluate the prevalence of N. salmonis in out-migrating juvenile hatchery and wild Chinook salmon Oncorhynchus tshawytscha and steelhead O. mykiss from the Snake River in the U.S. Pacific Northwest. To achieve this goal, we first addressed the following concerns about current molecular diagnostic tests for N. salmonis: (1) nonspecific amplification patterns by the published nested polymerase chain reaction (nPCR) test, (2) incomplete validation of the published quantitative PCR (qPCR) test, and (3) whether N. salmonis can be detected reliably from nonlethal samples. Here, we present an optimized nPCR protocol that eliminates nonspecific amplification. During validation of the published qPCR test, our laboratory developed a second qPCR test that targeted a different gene sequence and used different probe chemistry for comparison purposes. We simultaneously evaluated the two different qPCR tests for N. salmonis and found that both assays were highly specific, sensitive, and repeatable. The nPCR and qPCR tests had good overall concordance when DNA samples derived from both apparently healthy and clinically diseased hatchery rainbow trout were tested. Finally, we demonstrated that gill snips were a suitable tissue for nonlethal detection of N. salmonis DNA in juvenile salmonids. Monitoring of juvenile salmonid fish in the Snake River over a 3-year period revealed low prevalence of N. salmonis in hatchery and wild Chinook salmon and wild steelhead but significantly higher prevalence in hatchery-derived steelhead. Routine monitoring of N. salmonis is not performed for all hatchery steelhead populations. At present, the possible contribution of this pathogen to delayed mortality of steelhead has not been determined.

Duplex detection of the Mycobacterium tuberculosis complex and medically important non-tuberculosis mycobacteria by real-time PCR based on the rnpB gene.

PubMed

Abdeldaim, Guma; Svensson, Erik; Blomberg, Jonas; Herrmann, Björn

2016-11-01

A duplex real-time PCR based on the rnpB gene was developed for Mycobacterium spp. The assay was specific for the Mycobacterium tuberculosis complex (MTB) and also detected all 19 tested species of non-tuberculous mycobacteria (NTM). The assay was evaluated on 404 clinical samples: 290 respiratory samples and 114 from tissue and other non-respiratory body sites. M. tuberculosis was detected by culture in 40 samples and in 30 samples by the assay. The MTB assay showed a sensitivity similar to Roche Cobas Amplicor MTB-PCR (Roche Molecular Systems, Pleasanton, CA, USA). There were only nine samples with non-tuberculous mycobacteria detected by culture. Six of them were detected by the PCR assay. © 2016 APMIS. Published by John Wiley & Sons Ltd.

Optimization of the elution buffer and concentration method for detecting hepatitis E virus in swine liver using a nested reverse transcription-polymerase chain reaction and real-time reverse transcription-polymerase chain reaction.

PubMed

Son, Na Ry; Seo, Dong Joo; Lee, Min Hwa; Seo, Sheungwoo; Wang, Xiaoyu; Lee, Bog-Hieu; Lee, Jeong-Su; Joo, In-Sun; Hwang, In-Gyun; Choi, Changsun

2014-09-01

The aim of this study was to develop an optimal technique for detecting hepatitis E virus (HEV) in swine livers. Here, three elution buffers and two concentration methods were compared with respect to enhancing recovery of HEV from swine liver samples. Real-time reverse transcription-polymerase chain reaction (RT-PCR) and nested RT-PCR were performed to detect HEV RNA. When phosphate-buffered saline (PBS, pH 7.4) was used to concentrate HEV in swine liver samples using ultrafiltration, real-time RT-PCR detected HEV in 6 of the 26 samples. When threonine buffer was used to concentrate HEV using polyethylene glycol (PEG) precipitation and ultrafiltration, real-time RT-PCR detected HEV in 1 and 3 of the 26 samples, respectively. When glycine buffer was used to concentrate HEV using ultrafiltration and PEG precipitation, real-time RT-PCR detected HEV in 1 and 3 samples of the 26 samples, respectively. When nested RT-PCR was used to detect HEV, all samples tested negative regardless of the type of elution buffer or concentration method used. Therefore, the combination of real-time RT-PCR and ultrafiltration with PBS buffer was the most sensitive and reliable method for detecting HEV in swine livers. Copyright © 2014 Elsevier B.V. All rights reserved.

Comparison of real-time PCR methods for the detection of Naegleria fowleri in surface water and sediment.

PubMed

Streby, Ashleigh; Mull, Bonnie J; Levy, Karen; Hill, Vincent R

2015-05-01

Naegleria fowleri is a thermophilic free-living ameba found in freshwater environments worldwide. It is the cause of a rare but potentially fatal disease in humans known as primary amebic meningoencephalitis. Established N. fowleri detection methods rely on conventional culture techniques and morphological examination followed by molecular testing. Multiple alternative real-time PCR assays have been published for rapid detection of Naegleria spp. and N. fowleri. Foursuch assays were evaluated for the detection of N. fowleri from surface water and sediment. The assays were compared for thermodynamic stability, analytical sensitivity and specificity, detection limits, humic acid inhibition effects, and performance with seeded environmental matrices. Twenty-one ameba isolates were included in the DNA panel used for analytical sensitivity and specificity analyses. N. fowleri genotypes I and III were used for method performance testing. Two of the real-time PCR assays were determined to yield similar performance data for specificity and sensitivity for detecting N. fowleri in environmental matrices.

Comparison of real-time PCR methods for the detection of Naegleria fowleri in surface water and sediment

PubMed Central

Streby, Ashleigh; Mull, Bonnie J.; Levy, Karen

2015-01-01

Naegleria fowleri is a thermophilic free-living ameba found in freshwater environments worldwide. It is the cause of a rare but potentially fatal disease in humans known as primary amebic meningoencephalitis. Established N. fowleri detection methods rely on conventional culture techniques and morphological examination followed by molecular testing. Multiple alternative real-time PCR assays have been published for rapid detection of Naegleria spp. and N. fowleri. Four such assays were evaluated for the detection of N. fowleri from surface water and sediment. The assays were compared for thermodynamic stability, analytical sensitivity and specificity, detection limits, humic acid inhibition effects, and performance with seeded environmental matrices. Twenty-one ameba isolates were included in the DNA panel used for analytical sensitivity and specificity analyses. N. fowleri genotypes I and III were used for method performance testing. Two of the real-time PCR assays were determined to yield similar performance data for specificity and sensitivity for detecting N. fowleri in environmental matrices. PMID:25855343

Combined evaluation of commonly used techniques, including PCR, for diagnosis of mouse fur mites.

PubMed

Karlsson, Eleanor M; Pearson, Laura M; Kuzma, Kristen M; Burkholder, Tanya H

2014-01-01

Our study evaluated and compared the false-negative rates (FNR) of a wide array of fur-mite diagnostic tests, including 2 postmortem tests (pelt exam and sticky paper) and 3 antemortem tests (adhesive tape, fur pluck, and PCR). Past publications examining fur-mite diagnostic techniques primarily used paired comparisons, evaluating tests by their level of agreement with only one other test. However, different combinations or pairs of diagnostics are used in the different studies, making the results of these comparisons difficult to interpret across all available diagnostics. In the current study, mice from a conventionally maintained colony endemic for Myobia musculi were identified as positive based on at least one positive diagnostic test. From this pool of positive animals, the FNR of all tests were quantified. The PCR assay and the pelt exam performed the best, with 0% and 2% FNR respectively, whereas tape, fur-pluck, and sticky-paper tests showed 24%, 26%, and 36% FNR, respectively. Our study shows that for mice in a colony naturally infested with Myobia musculi, PCR testing can be used for reliable antemortem detection, and pelt exam performed by experienced examiners is reliable for postmortem detection.

Evaluation of a duplex reverse-transcription real-time PCR assay for the detection of encephalomyocarditis virus.

PubMed

Qin, Shaomin; Underwood, Darren; Driver, Luke; Kistler, Carol; Diallo, Ibrahim; Kirkland, Peter D

2018-06-01

We evaluated a fluorogenic probe-based assay for the detection of encephalomyocarditis virus (EMCV) by comparing a set of published primers and probe to a new set of primers and probe. The published reagents failed to amplify a range of Australian isolates and an Italian reference strain of EMCV. In contrast, an assay based on 2 new sets of primers and probes that were run in a duplex reverse-transcription real-time PCR (RT-rtPCR) worked well, with high amplification efficiency. The analytical sensitivity was ~100-fold higher than virus isolation in cell culture. The intra-assay variation was 0.21-4.90%. No cross-reactivity was observed with a range of other porcine viruses. One hundred and twenty-two clinical specimens were tested simultaneously by RT-rtPCR and virus isolation in cell culture; 72 specimens gave positive results by RT-rtPCR, and 63 of these were also positive by virus isolation. Of 245 archived cell culture isolates of EMCV that were tested in the RT-rtPCR, 242 samples were positive. The new duplex RT-rtPCR assay is a reliable tool for the detection of EMCV in clinical specimens and for use in epidemiologic investigations.

Quantitative fucK gene polymerase chain reaction on sputum and nasopharyngeal secretions to detect Haemophilus influenzae pneumonia.

PubMed

Abdeldaim, Guma M K; Strålin, Kristoffer; Olcén, Per; Blomberg, Jonas; Mölling, Paula; Herrmann, Björn

2013-06-01

A quantitative polymerase chain reaction (PCR) for the fucK gene was developed for specific detection of Haemophilus influenzae. The method was tested on sputum and nasopharyngeal aspirate (NPA) from 78 patients with community-acquired pneumonia (CAP). With a reference standard of sputum culture and/or serology against the patient's own nasopharyngeal isolate, H. influenzae etiology was detected in 20 patients. Compared with the reference standard, fucK PCR (using the detection limit 10(5) DNA copies/mL) on sputum and NPA showed a sensitivity of 95.0% (19/20) in both cases, and specificities of 87.9% (51/58) and 89.5% (52/58), respectively. In a receiver operating characteristic curve analysis, sputum fucK PCR was found to be significantly superior to sputum P6 PCR for detection of H. influenzae CAP. NPA fucK PCR was positive in 3 of 54 adult controls without respiratory symptoms. In conclusion, quantitative fucK real-time PCR provides a sensitive and specific identification of H. influenzae in respiratory secretions. Copyright © 2013 Elsevier Inc. All rights reserved.

The application of uniplex, duplex, and multiplex PCR for the absence of specified microorganism testing of pharmaceutical excipients and drug products.

PubMed

Ragheb, Suzan M; Yassin, Aymen S; Amin, Magdy A

2012-01-01

Notable progress has been made in methods that encourage the use of polymerase chain reaction (PCR) as a rapid and accurate tool in microbiological testing of pharmaceuticals. In this study, the detection of the four main specified microorganisms according to the pharmacopeial recommendations, Salmonella spp, Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus, was optimized in different pharmaceutical dosage forms and raw materials. Uniplex PCR was performed for the detection of each microorganism individually targeting the conserved region in each bacterial genome. Further optimizations were done to perform duplex and multiplex PCR assays considering relative concentrations of competitor primers used in the reaction. The uniplex PCR amplicons were successfully sequenced, confirming the conservation of used primers. Other validation parameters such as specificity, sensitivity, and robustness were examined closely. The method provides a high-throughput screening method to test different pharmaceutical preparations for specified microorganisms for the detection of microbiological contamination. Strict regulations govern the production of pharmaceutical products whether they are sterile or nonsterile. Certain official tests are carried out in microbiology testing laboratory in any pharmaceutical production facility to ensure the pharmaceuticals microbiological quality according to the standard pharmacopeial recommendations. Nonsterile products must be free of specified microorganisms that are used as a check for their quality. Topical preparations must be free of Pseudomonas aeruginosa and Staphylococcus aureus, and oral preparations must be free of Salmonella spp and Escherichia coli. Conventional microbiological methods are time-consuming, labor-intensive, and require long incubation times, resulting in delaying the release of the products. In this study, we tested and validated a polymerase chain reaction identification approach to detect indicator bacteria in pharmaceutical preparations. The method depends on amplification of certain conserved genes located in the four specified bacteria. The method is optimized to be carried out individually or collectively to detect all indicator bacteria in a single reaction in different forms of pharmaceutical products.

Final Report Nucleic Acid System - Hybrid PCR and Multiplex Assay Project Phase 2

DOE Office of Scientific and Technical Information (OSTI.GOV)

Koopman, R P; Langlois, R G; Nasarabadi, S

2002-04-17

This report covers phase 2 (year 2) of the Nucleic Acid System--Hybrid PCR and Multiplex Assay project. The objective of the project is to reduce to practice the detection and identification of biological warfare pathogens by the nucleic acid recognition technique of PCR (polymerase chain reaction) in a multiplex mode using flow cytometry. The Hybrid instrument consists of a flow-through PCR module capable of handling a multiplexed PCR assay, a hybridizing module capable of hybridizing multiplexed PCR amplicons and beads, and a flow cytometer module for bead-based identification, all controlled by a single computer. Multiplex immunoassay using bead-based Luminex flowmore » cytometry is available, allowing rapid screening for many agents. PCR is highly specific and complements and verifies immunoassay. It can also be multiplexed and detection provided using the bead-based Luminex flow cytometer. This approach allows full access to the speed and 100-fold multiplex capability of flow cytometry for rapid screening as well as the accuracy and specificity of PCR. This project has two principal activities: (1) Design, build and test a prototype hybrid PCR/flow cytometer with the basic capabilities for rapid, broad spectrum detection and identification, and (2) Develop and evaluate multiplex flow analysis assay protocols and reagents for the simultaneous detection of PCR products. This project requires not only building operationally functional instrumentation but also developing the chemical assays for detection of priority pathogens. This involves development and evaluation of multiplex flow analysis assay protocols and reagents for the simultaneous detection of PCR products.« less

Comparison of the Sensitivity of Laboratory Diagnostic Methods from a Well-Characterized Outbreak of Mumps in New York City in 2009

PubMed Central

Rosen, Jennifer B.; Doll, Margaret K.; McNall, Rebecca J.; McGrew, Marcia; Williams, Nobia; Lopareva, Elena N.; Barskey, Albert E.; Punsalang, Amado; Rota, Paul A.; Oleszko, William R.; Hickman, Carole J.; Zimmerman, Christopher M.; Bellini, William J.

2013-01-01

A mumps outbreak in upstate New York in 2009 at a summer camp for Orthodox Jewish boys spread into Orthodox Jewish communities in the Northeast, including New York City. The availability of epidemiologic information, including vaccination records and parotitis onset dates, allowed an enhanced analysis of laboratory methods for mumps testing. Serum and buccal swab samples were collected from 296 confirmed cases with onsets from September through December 2009. All samples were tested using the Centers for Disease Control and Prevention (CDC) capture IgM enzyme immunoassay (EIA) and a real-time reverse transcription-PCR (rRT-PCR) that targets the short hydrophobic gene. A subset of the samples (n = 205) was used to evaluate 3 commercial mumps IgM assays and to assess the sensitivity of using an alternative target gene (nucleoprotein) in the rRT-PCR protocol. Among 115 cases of mumps with 2 documented doses of measles, mumps, and rubella (MMR) vaccine, the CDC capture IgM EIA detected IgM in 51% of serum samples compared to 9% to 24% using three commercial IgM assays. The rRT-PCR that targeted the nucleoprotein gene increased RNA detection by 14% compared to that obtained with the original protocol. The ability to detect IgM improved when serum was collected 3 days or more after symptom onset, whereas sensitivity of RNA detection by rRT-PCR declined when buccal swabs were collected later than 2 days after onset. Selection of testing methods and timing of sample collection are important factors in the ability to confirm infection among vaccinated persons. These results reinforce the need to use virus detection assays in addition to serologic tests. PMID:23324519

Detection of enteroviruses and hepatitis a virus in water by consensus primer multiplex RT-PCR

PubMed Central

Li, Jun-Wen; Wang, Xin-Wei; Yuan, Chang-Qing; Zheng, Jin-Lai; Jin, Min; Song, Nong; Shi, Xiu-Quan; Chao, Fu-Huan

2002-01-01

AIM: To develop a rapid detection method of enteroviruses and Hepatitis A virus (HAV). METHODS: A one-step, single-tube consensus primers multiplex RT-PCR was developed to simultaneously detect Poliovirus, Coxsackie virus, Echovirus and HAV. A general upstream primer and a HAV primer and four different sets of primers (5 primers) specific for Poliovirus, Coxsacki evirus, Echovirus and HAV cDNA were mixed in the PCR mixture to reverse transcript and amplify the target DNA. Four distinct amplified DNA segments representing Poliovirus, Coxsackie virus, Echovirus and HAV were identified by gel electrophoresis as 589-, 671-, 1084-, and 1128 bp sequences, respectively. Semi-nested PCR was used to confirm the amplified products for each enterovirus and HAV. RESULTS: All four kinds of viral genome RNA were detected, and producing four bands which could be differentiated by the band size on the gel. To confirm the specificity of the multiplex PCR products, semi-nested PCR was performed. For all the four strains tested gave positive results. The detection sensitivity of multiplex PCR was similar to that of monoplex RT-PCR which was 24 PFU for Poliovrus, 21 PFU for Coxsackie virus, 60 PFU for Echovirus and 105 TCID50 for HAV. The minimum amount of enteric viral RNA detected by semi-nested PCR was equivalent to 2.4 PFU for Poliovrus, 2.1 PFU for Coxsackie virus, 6.0 PFU for Echovirus and 10.5 TCID50 for HAV. CONCLUSION: The consensus primers multiplex RT-PCR has more advantages over monoplex RT-PCR for enteric viruses detection, namely, the rapid turnaround time and cost effectiveness. PMID:12174381

Detection of Echinococcus multilocularis by MC-PCR: evaluation of diagnostic sensitivity and specificity without gold standard

PubMed Central

Wahlström, Helene; Comin, Arianna; Isaksson, Mats; Deplazes, Peter

2016-01-01

Introduction A semi-automated magnetic capture probe-based DNA extraction and real-time PCR method (MC-PCR), allowing for a more efficient large-scale surveillance of Echinococcus multilocularis occurrence, has been developed. The test sensitivity has previously been evaluated using the sedimentation and counting technique (SCT) as a gold standard. However, as the sensitivity of the SCT is not 1, test characteristics of the MC-PCR was also evaluated using latent class analysis, a methodology not requiring a gold standard. Materials and methods Test results, MC-PCR and SCT, from a previous evaluation of the MC-PCR using 177 foxes shot in the spring (n=108) and autumn 2012 (n=69) in high prevalence areas in Switzerland were used. Latent class analysis was used to estimate the test characteristics of the MC-PCR. Although it is not the primary aim of this study, estimates of the test characteristics of the SCT were also obtained. Results and discussion This study showed that the sensitivity of the MC-PCR was 0.88 [95% posterior credible interval (PCI) 0.80–0.93], which was not significantly different than the SCT, 0.83 (95% PCI 0.76–0.88), which is currently considered as the gold standard. The specificity of both tests was high, 0.98 (95% PCI 0.94–0.99) for the MC-PCR and 0.99 (95% PCI 0.99–1) for the SCT. In a previous study, using fox scats from a low prevalence area, the specificity of the MC-PCR was higher, 0.999% (95% PCI 0.997–1). One reason for the lower estimate of the specificity in this study could be that the MC-PCR detects DNA from infected but non-infectious rodents eaten by foxes. When using MC-PCR in low prevalence areas or areas free from the parasite, a positive result in the MC-PCR should be regarded as a true positive. Conclusion The sensitivity of the MC-PCR (0.88) was comparable to the sensitivity of SCT (0.83). PMID:26968153

[Investigation of Coxiella burnetii contamination in commercial milk and PCR method for the detection of C. burnetii in egg].

PubMed

Hirai, Akihiko; Kaneko, Seiji; Nakama, Akiko; Ishizaki, Naoto; Odagiri, Megumi; Kai, Akemi; Sadamasu, Kenji; Shinkai, Takayuki; Yano, Kazuyoshi; Morozumi, Satoshi

2005-06-01

A total of 244 milk samples collected from supermarkets in Tokyo were examined for contamination with Coxiella burnetii. C. burnetii DNA was detected in 131 (53.7%) of the samples by nested PCR. PCR-positive samples were injected into immunosuppressed A/J strain mice. Of the 22 PCR-positive milk samples tested, none resulted in isolation of C. burnetii from the mice. Heat-treatment was sufficient to inactivate C. burnetii in commercial milk. In addition, a PCR detection method for C. burnetii in chicken egg was developed. Egg yolk was added to an equal volume of 1 mol/L of NaCl phosphate buffer and homogenized for removal of protein and lipid. After centrifugal separation, the supernatant was removed, and template DNA in the precipitate was extracted using SDS, proteinase K and NaI. Using such prepared samples, 3.2 x 10(1) C. burnetii particles in 1 g of egg yolk could be detected by nested PCR. All of 200 chicken egg samples collected from supermarkets in Tokyo were negative for C. burnetii by the nested PCR method.

Detection and quantification of Renibacterium salmoninarum DNA in salmonid tissues by real-time quantitative polymerase chain reaction analysis

USGS Publications Warehouse

Chase, D.M.; Elliott, D.G.; Pascho, R.J.

2006-01-01

Renibacterium salmoninarum is an important salmonid pathogen that is difficult to culture. We developed and assessed a real-time, quantitative, polymerase chain reaction (qPCR) assay for the detection and enumeration of R. salmoninarum. The qPCR is based on TaqMan technology and amplifies a 69-base pair (bp) region of the gene encoding the major soluble antigen (MSA) of R. salmoninarum. The qPCR assay consistently detected as few as 5 R. salmoninarum cells per reaction in kidney tissue. The specificity of the qPCR was confirmed by testing the DNA extracts from a panel of microorganisms that were either common fish pathogens or reported to cause false-positive reactions in the enzyme-linked immunosorbent assay (ELISA). Kidney samples from 38 juvenile Chinook salmon (Oncorhynchus tshawytscha) in a naturally infected population were examined by real-time qPCR, a nested PCR, and ELISA, and prevalences of R. salmoninarum detected were 71, 66, and 71%, respectively. The qPCR should be a valuable tool for evaluating the R. salmoninarum infection status of salmonids.

Recombinase Polymerase Amplification Assay-A Simple, Fast and Cost-Effective Alternative to Real Time PCR for Specific Detection of Feline Herpesvirus-1.

PubMed

Wang, Jianchang; Liu, Libing; Wang, Jinfeng; Sun, Xiaoxia; Yuan, Wanzhe

2017-01-01

Feline herpesvirus 1 (FHV-1), an enveloped dsDNA virus, is one of the major pathogens of feline upper respiratory tract disease (URTD) and ocular disease. Currently, polymerase chain reaction (PCR) remains the gold standard diagnostic tool for FHV-1 infection but is relatively expensive, requires well-equipped laboratories and is not suitable for field tests. Recombinase polymerase amplification (RPA), an isothermal gene amplification technology, has been explored for the molecular diagnosis of infectious diseases. In this study, an exo-RPA assay for FHV-1 detection was developed and validated. Primers targeting specifically the thymidine kinase (TK) gene of FHV-1 were designed. The RPA reaction was performed successfully at 39°C and the results were obtained within 20 min. Using different copy numbers of recombinant plasmid DNA that contains the TK gene as template, we showed the detection limit of exo-RPA was 102 copies DNA/reaction, the same as that of real time PCR. The exo-RPA assay did not cross-detect feline panleukopenia virus, feline calicivirus, bovine herpesvirus-1, pseudorabies virus or chlamydia psittaci, a panel of pathogens important in feline URTD or other viruses in Alphaherpesvirinae, demonstrating high specificity. The assay was validated by testing 120 nasal and ocular conjunctival swabs of cats, and the results were compared with those obtained with real-time PCR. Both assays provided the same testing results in the clinical samples. Compared with real time PCR, the exo-RPA assay uses less-complex equipment that is portable and the reaction is completed much faster. Additionally, commercial RPA reagents in vacuum-sealed pouches can tolerate temperatures up to room temperature for days without loss of activity, suitable for shipment and storage for field tests. Taken together, the exo-RPA assay is a simple, fast and cost-effective alternative to real time PCR, suitable for use in less advanced laboratories and for field detection of FHV-1 infection.

Recombinase Polymerase Amplification Assay—A Simple, Fast and Cost-Effective Alternative to Real Time PCR for Specific Detection of Feline Herpesvirus-1

PubMed Central

Wang, Jianchang; Liu, Libing; Wang, Jinfeng; Sun, Xiaoxia; Yuan, Wanzhe

2017-01-01

Feline herpesvirus 1 (FHV-1), an enveloped dsDNA virus, is one of the major pathogens of feline upper respiratory tract disease (URTD) and ocular disease. Currently, polymerase chain reaction (PCR) remains the gold standard diagnostic tool for FHV-1 infection but is relatively expensive, requires well-equipped laboratories and is not suitable for field tests. Recombinase polymerase amplification (RPA), an isothermal gene amplification technology, has been explored for the molecular diagnosis of infectious diseases. In this study, an exo-RPA assay for FHV-1 detection was developed and validated. Primers targeting specifically the thymidine kinase (TK) gene of FHV-1 were designed. The RPA reaction was performed successfully at 39°C and the results were obtained within 20 min. Using different copy numbers of recombinant plasmid DNA that contains the TK gene as template, we showed the detection limit of exo-RPA was 102 copies DNA/reaction, the same as that of real time PCR. The exo-RPA assay did not cross-detect feline panleukopenia virus, feline calicivirus, bovine herpesvirus-1, pseudorabies virus or chlamydia psittaci, a panel of pathogens important in feline URTD or other viruses in Alphaherpesvirinae, demonstrating high specificity. The assay was validated by testing 120 nasal and ocular conjunctival swabs of cats, and the results were compared with those obtained with real-time PCR. Both assays provided the same testing results in the clinical samples. Compared with real time PCR, the exo-RPA assay uses less-complex equipment that is portable and the reaction is completed much faster. Additionally, commercial RPA reagents in vacuum-sealed pouches can tolerate temperatures up to room temperature for days without loss of activity, suitable for shipment and storage for field tests. Taken together, the exo-RPA assay is a simple, fast and cost-effective alternative to real time PCR, suitable for use in less advanced laboratories and for field detection of FHV-1 infection. PMID:28045956

Loop-mediated isothermal amplification assay for detection of Haemophilus influenzae type b in cerebrospinal fluid.

PubMed

Kim, Dong Wook; Kilgore, Paul Evan; Kim, Eun Jin; Kim, Soon Ae; Anh, Dang Duc; Seki, Mitsuko

2011-10-01

Haemophilus influenzae type b (Hib) is one of the leading causes of meningitis in developing countries. To establish and evaluate a novel loop-mediated isothermal amplification (LAMP) assay for Hib, we designed a LAMP primer set targeting the Hib-specific capsulation locus. LAMP detected 10 copies of purified DNA in a 60-min reaction. This indicated that the detection limit of LAMP was >100-fold lower than the detection limits of both a PCR for the detection of bexA and a nested PCR for Hib (Hib PCR). No H. influenzae, other than Hib or control bacteria, was detected. Linear determination ranged from 10 to 1,000,000 microorganisms per reaction mixture using real-time turbidimetry. We evaluated the Hib LAMP assay using a set of 52 randomly selected cerebrospinal fluid (CSF) specimens obtained from children with suspected meningitis. For comparison, the CSF specimens were tested using a conventional Hib PCR assay. Hib was detected in 30 samples using LAMP and in 22 samples using the Hib PCR assay. The Hib PCR showed a clinical sensitivity of 73.3% and a clinical specificity of 100% relative to the Hib LAMP assay. These results suggest that further development and evaluation of the Hib LAMP will enhance the global diagnostic capability for Hib detection.

Comparison between ICT and PCR for diagnosis of Chlamydia trachomatis.

PubMed

Khan, E R; Hossain, M A; Paul, S K; Mahmud, C; Hasan, M M; Rahman, M M; Nahar, K; Kubayashi, N

2012-04-01

Chlamydia trachomatis is an obligate intracellular gram-negative bacterium which is the most prevalent cause of bacterial sexually transmitted infections (STI). The present study was carried to diagnose genital Chlamydia trachomatis infection among women of reproductive age, attending Mymensingh Medical College Hospital, during July 2009 to June 2010 by Immunochromatographic test (ICT) and Polymerase chain reaction (PCR). A total of 70 females were included in this study. Out of 70 cases 56 were symptomatic and 14 asymptomatic. Endocervical swabs were collected from each of the cases and examined by Immunochromatographic test (ICT) for antigen detection and Polymerase chain reaction (PCR) for detection of endogenous plasmid-based nucleic acid. A total 29(41.4%) of the cases were found positive for C. trachomatis either by ICT or PCR. Of the 56 symptomatic cases, 19(33.9%) were found ICT positive and 17(30.4%) were PCR positive. Among 14 asymptomatic females, 2(14.3%) were ICT positive and none were PCR positive. Though PCR is highly sensitive but a total of twelve cases were found ICT positive but PCR negative. It may be due to presence of plasmid deficient strain of C trachomatis which could be amplified by ompA based (Chromosomal gene) multiplex PCR.

Use of Competitive PCR to Detect and Quantify Haplosporidium nelsoni Infection (MSX disease) in the Eastern Oyster (Crassostrea virginica).

PubMed

Day, J Michael; Franklin, Dean E.; Brown, Bonnie L.

2000-09-01

This study was undertaken to develop a quantitative polymerase chain reaction assay that would improve the utility of PCR for detecting Haplosporidium nelsoni (MSX), a serious parasite of the eastern oyster Crassostrea virginica. A competitive PCR sequence was generated from the H. nelsoni small subunit ribosomal DNA fragment, originally described by Stokes and colleagues, that was amplified by the same PCR primers and had similar amplification performance. Assays performed using competitor dilutions ranging from 0.05 to 500 pg/µl DNA were used to test oyster samples designated using histological techniques as having "light" or "heavy" MSX infections. Visual diagnoses were confirmed equally well with three methods: densitometry of ethidium-bromide-stained agarose, densitometry of SYBRGreen-stained polyacrylamide gels, and analysis by GeneScan 3.0 of fluorescent products detected in ultrathin gels. Oysters diagnosed as negative for MSX tested as negative or light by PCR. Oysters with light MSX infections generally had less than 5 pg/µl infectious DNA. Oysters with heavy infections generally corresponded to 5 pg/µl or greater competitor dilutions.

Recent sequence variation in probe binding site affected detection of respiratory syncytial virus group B by real-time RT-PCR.

PubMed

Kamau, Everlyn; Agoti, Charles N; Lewa, Clement S; Oketch, John; Owor, Betty E; Otieno, Grieven P; Bett, Anne; Cane, Patricia A; Nokes, D James

2017-03-01

Direct immuno-fluorescence test (IFAT) and multiplex real-time RT-PCR have been central to RSV diagnosis in Kilifi, Kenya. Recently, these two methods showed discrepancies with an increasing number of PCR undetectable RSV-B viruses. Establish if mismatches in the primer and probe binding sites could have reduced real-time RT-PCR sensitivity. Nucleoprotein (N) and glycoprotein (G) genes were sequenced for real-time RT-PCR positive and negative samples. Primer and probe binding regions in N gene were checked for mismatches and phylogenetic analyses done to determine molecular epidemiology of these viruses. New primers and probe were designed and tested on the previously real-time RT-PCR negative samples. N gene sequences revealed 3 different mismatches in the probe target site of PCR negative, IFAT positive viruses. The primers target sites had no mismatches. Phylogenetic analysis of N and G genes showed that real-time RT-PCR positive and negative samples fell into distinct clades. Newly designed primers-probe pair improved detection and recovered previous PCR undetectable viruses. An emerging RSV-B variant is undetectable by a quite widely used real-time RT-PCR assay due to polymorphisms that influence probe hybridization affecting PCR accuracy. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

A PCR method for the detection and differentiation of Lentinus edodes and Trametes versicolor in defined-mixed cultures used for wastewater treatment.

PubMed

García-Mena, Jaime; Cano-Ramirez, Claudia; Garibay-Orijel, Claudio; Ramirez-Canseco, Sergio; Poggi-Varaldo, Héctor M

2005-06-01

A PCR-based method for the quantitative detection of Lentinus edodes and Trametes versicolor, two ligninolytic fungi applied for wastewater treatment and bioremediation, was developed. Genomic DNA was used to optimize a PCR method targeting the conserved copper-binding sequence of laccase genes. The method allowed the quantitative detection and differentiation of these fungi in single and defined-mixed cultures after fractionation of the PCR products by electrophoresis in agarose gels. Amplified products of about 150 bp for L. edodes, and about 200 bp for T. versicolor were purified and cloned. The PCR method showed a linear detection response in the 1.0 microg-1 ng range. The same method was tested with genomic DNA from a third fungus (Phanerochaete chrysosporium), yielding a fragment of about 400 bp. Southern-blot and DNA sequence analysis indicated that a specific PCR product was amplified from each genome, and that these corresponded to sequences of laccase genes. This PCR protocol permits the detection and differentiation of three ligninolytic fungi by amplifying DNA fragments of different sizes using a single pair of primers, without further enzymatic restriction of the PCR products. This method has potential use in the monitoring, evaluation, and improvement of fungal cultures used in wastewater treatment processes.

Detection of Helicobacter pylori by Real-Time PCR for 16s rRNA in Stools of NonInfected Healthy Children, Using ELISA Antigen Stool Test as the Gold Standard.

PubMed

George, Sergio; Mamani, Nora; Lucero, Yalda; Torres, Juan Pablo; Farfán, Mauricio; Lagomarcino, Anne J; Orellana, Andrea; O'Ryan, Miguel

2016-12-01

We previously detected Helicobacter pylori infection by stool antigen ELISA assay in 33-41% of asymptomatic Chilean children between 2-3 years of age, of which 11-20% had a transient infection and 21-22% a persistent infection. A total of 88% of ELISA-positive samples were also rtPCR positive, while 37/133 (33%) of ELISA-negative stool samples were rtPCR positive. The significance of a ELISA-negative/rtPCR-positive sample requires clarification. We aimed to determine whether rtPCR is able to detect persistent infections not detected by ELISA. We selected 36 children with an ELISA-negative/rtPCR-positive stool sample, of which 25 were never H. pylori infected according to ELISA, and 11 had a transient infection with an ELISA-positive sample before or after the discordant sample. At least two additional consecutive ELISA-negative samples per child were tested in duplicate by rtPCR for the 16s rRNA gene. A total of 14 of 78 (17.9%) rtPCR reactions were positive, but only 4/78 (5.1%) were positive in both duplicates, representing a total of 3/36 (8.3%) children with an additional rtPCR-positive sample, only one of whom was persistently negative by ELISA. One child with a transient infection had two positive rtPCR reactions despite negative ELISA samples. In H. pylori noninfected or transiently infected children, as determined by stool ELISA, additional ELISA-negative/rtPCR-positive stool samples were found in 8.3% of children, but a possible persistent infection was only identified in 2.7% of children. Thus, the characterization of infection dynamics in children is not being misrepresented by application of stool ELISA. Furthermore, rtPCR does not significantly improve dynamic characterization. © 2016 John Wiley & Sons Ltd.

Evaluation of a Real-Time PCR Kit for Detecting Escherichia coli O157 in Bovine Fecal Samples

PubMed Central

Bono, James L.; Keen, James E.; Miller, Laura C.; Fox, James M.; Chitko-McKown, Carol G.; Heaton, Michael P.; Laegreid, William W.

2004-01-01

A commercially available real-time, rapid PCR test was evaluated for its ability to detect Escherichia coli O157. Both the sensitivity and specificity of the assay were 99% for isolates in pure culture. The assay detected 1 CFU of E. coli O157:H7 g−1 in artificially inoculated bovine feces following enrichment. PMID:15006817

Value of Different Assays for Detection of Human Cytomegalovirus (HCMV) in Predicting the Development of HCMV Disease in Human Immunodeficiency Virus-Infected Patients

PubMed Central

Blank, Brian S. N.; Meenhorst, Pieter L.; Mulder, Jan Willem; Weverling, Gerrit Jan; Putter, Hein; Pauw, Wouter; van Dijk, Willemien C.; Smits, Paul; Lie-A-Ling, Sonja; Reiss, Peter; Lange, Joep M. A.

2000-01-01

In the present prospective study, five blood tests for detection of human cytomegalovirus (HCMV), nucleic acid sequence-based amplification (NASBA) for detection of early (immediate-early antigen) and late (pp67) mRNA, PCR for detection of HCMV DNA (DNA PCR), culture, and pp65 antigenemia assay, and culture and DNA PCR of urine and throat swab specimens were compared for their abilities to predict the development of disease caused by HCMV (HCMV disease). Of 101 human immunodeficiency virus (HIV)-infected patients with ≤100 CD4+ lymphocytes per mm3, 25 patients developed HCMV disease. The pp65 antigenemia assay (sensitivity, 50%; specificity, 89%) and DNA PCR of blood (sensitivity, 69%; specificity, 75%) were most accurate in predicting the development of HCMV disease within the next 12 months. Both blood culture and late pp67 mRNA NASBA had high specificities (91 and 90%, respectively) but low sensitivities (25 and 13%, respectively). The sensitivities of urine culture, DNA PCR, throat swab specimen culture, DNA PCR, and NASBA of blood for detection of the immediate-early antigen were 73, 87, 53, 67, and 63%, respectively, and the specificities were 58, 46, 76, 60, and 72%, respectively. The positive predictive values of all tests however, were low and did not exceed 50%. In conclusion, virological screening by these qualitative assays for detection of HCMV is of limited value for prediction of the development of HCMV disease in HIV-infected patients. PMID:10655346

Development of strain-specific PCR primers for quantitative detection of Bacillus mesentericus strain TO-A in human feces.

PubMed

Sato, Naoki; Seo, Genichiro; Benno, Yoshimi

2014-01-01

Strain-specific polymerase chain reaction (PCR) primers for detection of Bacillus mesentericus strain TO-A (BM TO-A) were developed. The randomly amplified polymorphic DNA (RAPD) technique was used to produce potential strain-specific markers. A 991-bp RAPD marker found to be strain-specific was sequenced, and two primer pairs specific to BM TO-A were constructed based on this sequence. In addition, we explored a more specific DNA region using inverse PCR, and designed a strain-specific primer set for use in real-time quantitative PCR (qPCR). These primer pairs were tested against 25 Bacillus subtilis strains and were found to be strain-specific. After examination of the detection limit and linearity of detection of BM TO-A in feces, the qPCR method and strain-specific primers were used to quantify BM TO-A in the feces of healthy volunteers who had ingested 3×10(8) colony forming unit (CFU) of BM TO-A per day in tablets. During the administration period, BM TO-A was detected in the feces of all 24 subjects, and the average number of BM TO-A detected using the culture method and qPCR was about 10(4.8) and 10(5.8) cells per gram of feces, respectively. Using the qPCR method, BM TO-A was detected in the feces of half of the subjects 3 d after withdrawal, and was detected in the feces of only one subject 1 week after withdrawal. These results suggest that the qPCR method using BM TO-A strain-specific primers is useful for the quantitative detection of this strain in feces.

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. Part III. Virus Detection by RT-qPCR

EPA Science Inventory

EPA Method 1615 measures enteroviruses and noroviruses present in environmental and drinking waters. The viral ribonucleic acid (RNA) from water sample concentrates is extracted and tested for enterovirus and norovirus RNA using reverse transcription-quantitative PCR (RT-qPCR). V...

DNA Differential Diagnosis of Taeniasis and Cysticercosis by Multiplex PCR

PubMed Central

Yamasaki, Hiroshi; Allan, James C.; Sato, Marcello Otake; Nakao, Minoru; Sako, Yasuhito; Nakaya, Kazuhiro; Qiu, Dongchuan; Mamuti, Wulamu; Craig, Philip S.; Ito, Akira

2004-01-01

Multiplex PCR was established for differential diagnosis of taeniasis and cysticercosis, including their causative agents. For identification of the parasites, multiplex PCR with cytochrome c oxidase subunit 1 gene yielded evident differential products unique for Taenia saginata and Taenia asiatica and for American/African and Asian genotypes of Taenia solium with molecular sizes of 827, 269, 720, and 984 bp, respectively. In the PCR-based detection of tapeworm carriers using fecal samples, the diagnostic markers were detected from 7 of 14 and 4 of 9 T. solium carriers from Guatemala and Indonesia, respectively. Test sensitivity may have been reduced by the length of time (up to 12 years) that samples were stored and/or small sample volumes (ca. 30 to 50 mg). However, the diagnostic markers were detected by nested PCR in five worm carriers from Guatemalan cases that were found to be negative by multiplex PCR. It was noteworthy that a 720 bp-diagnostic marker was detected from a T. solium carrier who was egg-free, implying that it is possible to detect worm carriers and treat before mature gravid proglottids are discharged. In contrast to T. solium carriers, 827-bp markers were detected by multiplex PCR in all T. saginata carriers. The application of the multiplex PCR would be useful not only for surveillance of taeniasis and cysticercosis control but also for the molecular epidemiological survey of these cestode infections. PMID:14766815

Ultra-Sensitive Detection of Plasmodium falciparum by Amplification of Multi-Copy Subtelomeric Targets

PubMed Central

Hofmann, Natalie; Mwingira, Felista; Shekalaghe, Seif; Robinson, Leanne J.; Mueller, Ivo; Felger, Ingrid

2015-01-01

Background Planning and evaluating malaria control strategies relies on accurate definition of parasite prevalence in the population. A large proportion of asymptomatic parasite infections can only be identified by surveillance with molecular methods, yet these infections also contribute to onward transmission to mosquitoes. The sensitivity of molecular detection by PCR is limited by the abundance of the target sequence in a DNA sample; thus, detection becomes imperfect at low densities. We aimed to increase PCR diagnostic sensitivity by targeting multi-copy genomic sequences for reliable detection of low-density infections, and investigated the impact of these PCR assays on community prevalence data. Methods and Findings Two quantitative PCR (qPCR) assays were developed for ultra-sensitive detection of Plasmodium falciparum, targeting the high-copy telomere-associated repetitive element 2 (TARE-2, ∼250 copies/genome) and the var gene acidic terminal sequence (varATS, 59 copies/genome). Our assays reached a limit of detection of 0.03 to 0.15 parasites/μl blood and were 10× more sensitive than standard 18S rRNA qPCR. In a population cross-sectional study in Tanzania, 295/498 samples tested positive using ultra-sensitive assays. Light microscopy missed 169 infections (57%). 18S rRNA qPCR failed to identify 48 infections (16%), of which 40% carried gametocytes detected by pfs25 quantitative reverse-transcription PCR. To judge the suitability of the TARE-2 and varATS assays for high-throughput screens, their performance was tested on sample pools. Both ultra-sensitive assays correctly detected all pools containing one low-density P. falciparum–positive sample, which went undetected by 18S rRNA qPCR, among nine negatives. TARE-2 and varATS qPCRs improve estimates of prevalence rates, yet other infections might still remain undetected when absent in the limited blood volume sampled. Conclusions Measured malaria prevalence in communities is largely determined by the sensitivity of the diagnostic tool used. Even when applying standard molecular diagnostics, prevalence in our study population was underestimated by 8% compared to the new assays. Our findings highlight the need for highly sensitive tools such as TARE-2 and varATS qPCR in community surveillance and for monitoring interventions to better describe malaria epidemiology and inform malaria elimination efforts. PMID:25734259

Evaluation of multiplex real-time PCR for detection of Haemophilus ducreyi, Treponema pallidum, herpes simplex virus type 1 and 2 in the diagnosis of genital ulcer disease in the Rakai District, Uganda.

PubMed

Suntoke, T R; Hardick, A; Tobian, A A R; Mpoza, B; Laeyendecker, O; Serwadda, D; Opendi, P; Gaydos, C A; Gray, R H; Wawer, M J; Quinn, T C; Reynolds, S J

2009-04-01

To develop a real-time PCR assay that reliably and accurately detects the predominant sexually transmitted aetiological agents of genital ulcer disease (GUD) (Haemophilus ducreyi, Treponema pallidum and herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2)) and to assess the use of real-time PCR diagnostic testing in a rural African field site. Two multiplex real-time PCR reactions were used to detect H ducreyi/and HSV-1/HSV-2 in ulcer swabs from 100 people with symptomatic genital ulcers in rural Rakai, Uganda. Results were compared with syphilis, HSV-1 and HSV-2 serology. Of 100 GUD samples analysed from 43 HIV positive and 57 HIV negative individuals, 71% were positive for one or more sexually transmitted infection (STI) pathogens by real-time PCR (61% for HSV-2, 5% for T pallidum, 3% for HSV-1, 1% for H ducreyi and 1% for dual H ducreyi/HSV-2). The frequency of HSV in genital ulcers was 56% (32/57) in HIV negative individuals and 77% (33/43) in HIV positive individuals (p = 0.037). Assay reproducibility was evaluated by repeat PCR testing in the USA with 96% agreement (kappa = 0.85). STI pathogens were detected in the majority of GUD swab samples from symptomatic patients in Rakai, Uganda, by real-time PCR. HSV-2 was the predominant cause of genital ulcers. Real-time PCR technology can provide sensitive, rapid and reproducible evaluation of GUD aetiology in a resource-limited setting.

High-throughput multiplexed T-cell-receptor excision circle quantitative PCR assay with internal controls for detection of severe combined immunodeficiency in population-based newborn screening.

PubMed

Gerstel-Thompson, Jacalyn L; Wilkey, Jonathan F; Baptiste, Jennifer C; Navas, Jennifer S; Pai, Sung-Yun; Pass, Kenneth A; Eaton, Roger B; Comeau, Anne Marie

2010-09-01

Real-time quantitative PCR (qPCR) targeting a specific marker of functional T cells, the T-cell-receptor excision circle (TREC), detects the absence of functional T cells and has a demonstrated clinical validity for detecting severe combined immunodeficiency (SCID) in infants. There is need for a qPCR TREC assay with an internal control to monitor DNA quality and the relative cellular content of the particular dried blood spot punch sampled in each reaction. The utility of the qPCR TREC assay would also be far improved if more tests could be performed on the same newborn screening sample. We approached the multiplexing of qPCR for TREC by attenuating the reaction for the reference gene, with focus on maintaining tight quality assurance for reproducible slopes and for prevention of sample-to-sample cross contamination. Statewide newborn screening for SCID using the multiplexed assay was implemented, and quality-assurance data were recorded. The multiplex qPCR TREC assay showed nearly 100% amplification efficiency for each of the TREC and reference sequences, clinical validity for multiple forms of SCID, and an analytic limit of detection consistent with prevention of contamination. The eluate and residual ghost from a 3.2-mm dried blood spot could be used as source material for multiplexed immunoassays and multiplexed DNA tests (Multiplex Plus), with no disruption to the multiplex TREC qPCR. Population-based SCID newborn screening programs should consider multiplexing for quality assurance purposes. Potential benefits of using Multiplex Plus include the ability to perform multianalyte profiling.

Evaluation of PCR Systems for Field Screening of Bacillus anthracis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ozanich, Richard M.; Colburn, Heather A.; Victry, Kristin D.

There is little published data on the performance of hand-portable polymerase chain reaction (PCR) instruments that could be used by first responders to determine if a suspicious powder contains a potential biothreat agent. We evaluated five commercially available hand-portable PCR instruments for detection of Bacillus anthracis (Ba). We designed a cost-effective, statistically-based test plan that allows instruments to be evaluated at performance levels ranging from 0.85-0.95 lower confidence bound (LCB) on the probability of detection (POD) at confidence levels of 80-95%. We assessed specificity using purified genomic DNA from 13 Ba strains and 18 Bacillus near neighbors, interference with 22more » common hoax powders encountered in the field, and PCR inhibition when Ba spores were spiked into these powders. Our results indicated that three of the five instruments achieved >0.95 LCB on the POD with 95% confidence at test concentrations of 2,000 genome equivalents/mL (comparable to 2,000 spores/mL), displaying more than sufficient sensitivity for screening suspicious powders. These instruments exhibited no false positive results or PCR inhibition with common hoax powders, and reliably detected Ba spores spiked into common hoax powders, though some issues with instrument controls were observed. Our testing approach enables efficient instrument performance testing to a statistically rigorous and cost-effective test plan to generate performance data that will allow users to make informed decisions regarding the purchase and use of biodetection equipment in the field.« less

Comparison of various primer sets for detection of Toxoplasma gondii by polymerase chain reaction in fetal tissues from naturally aborted foxes.

PubMed

Smielewska-Loś, E

2003-01-01

Tissues from 4 aborted polar foxes (3 samples of brain and 4 samples of liver) were selected for Toxoplasma gondii PCR assay. Positive results of serological tests of mothers and immunofluorescence test (IFT) of fetal organ smears were the criteria of sample selection. Five sets of primers designed from B1 gene and ITS1 sequences of T. gondii were used for detection of the parasite in fetal fox tissues. All used primer sets successfully amplified T. gondii DNA in PCR from organs which were positive by IFT. Single tube nested PCR also showed positive result from a sample negative by IFT, but this product was not confirmed. The studies showed usefullness of PCR for routine diagnosis of toxoplasmosis in carnivores.

Molecular method for detection of total coliforms in drinking water samples.

PubMed

Maheux, Andrée F; Boudreau, Dominique K; Bisson, Marc-Antoine; Dion-Dupont, Vanessa; Bouchard, Sébastien; Nkuranga, Martine; Bergeron, Michel G; Rodriguez, Manuel J

2014-07-01

This work demonstrates the ability of a bacterial concentration and recovery procedure combined with three different PCR assays targeting the lacZ, wecG, and 16S rRNA genes, respectively, to detect the presence of total coliforms in 100-ml samples of potable water (presence/absence test). PCR assays were first compared to the culture-based Colilert and MI agar methods to determine their ability to detect 147 coliform strains representing 76 species of Enterobacteriaceae encountered in fecal and environmental settings. Results showed that 86 (58.5%) and 109 (74.1%) strains yielded a positive signal with Colilert and MI agar methods, respectively, whereas the lacZ, wecG, and 16S rRNA PCR assays detected 133 (90.5%), 111 (75.5%), and 146 (99.3%) of the 147 total coliform strains tested. These assays were then assessed by testing 122 well water samples collected in the Québec City region of Canada. Results showed that 97 (79.5%) of the samples tested by culture-based methods and 95 (77.9%), 82 (67.2%), and 98 (80.3%) of samples tested using PCR-based methods contained total coliforms, respectively. Consequently, despite the high genetic variability of the total coliform group, this study demonstrated that it is possible to use molecular assays to detect total coliforms in potable water: the 16S rRNA molecular assay was shown to be as efficient as recommended culture-based methods. This assay might be used in combination with an Escherichia coli molecular assay to assess drinking water quality. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Molecular Method for Detection of Total Coliforms in Drinking Water Samples

PubMed Central

Boudreau, Dominique K.; Bisson, Marc-Antoine; Dion-Dupont, Vanessa; Bouchard, Sébastien; Nkuranga, Martine; Bergeron, Michel G.; Rodriguez, Manuel J.

2014-01-01

This work demonstrates the ability of a bacterial concentration and recovery procedure combined with three different PCR assays targeting the lacZ, wecG, and 16S rRNA genes, respectively, to detect the presence of total coliforms in 100-ml samples of potable water (presence/absence test). PCR assays were first compared to the culture-based Colilert and MI agar methods to determine their ability to detect 147 coliform strains representing 76 species of Enterobacteriaceae encountered in fecal and environmental settings. Results showed that 86 (58.5%) and 109 (74.1%) strains yielded a positive signal with Colilert and MI agar methods, respectively, whereas the lacZ, wecG, and 16S rRNA PCR assays detected 133 (90.5%), 111 (75.5%), and 146 (99.3%) of the 147 total coliform strains tested. These assays were then assessed by testing 122 well water samples collected in the Québec City region of Canada. Results showed that 97 (79.5%) of the samples tested by culture-based methods and 95 (77.9%), 82 (67.2%), and 98 (80.3%) of samples tested using PCR-based methods contained total coliforms, respectively. Consequently, despite the high genetic variability of the total coliform group, this study demonstrated that it is possible to use molecular assays to detect total coliforms in potable water: the 16S rRNA molecular assay was shown to be as efficient as recommended culture-based methods. This assay might be used in combination with an Escherichia coli molecular assay to assess drinking water quality. PMID:24771030

Utility of pooled urine specimens for detection of Chlamydia trachomatis and Neisseria gonorrhoeae in men attending public sexually transmitted infection clinics in Mumbai, India, by PCR.

PubMed

Lindan, Christina; Mathur, Meenakshi; Kumta, Sameer; Jerajani, Hermangi; Gogate, Alka; Schachter, Julius; Moncada, Jeanne

2005-04-01

Pooling urogenital specimens for the detection of Chlamydia trachomatis and Neisseria gonorrhoeae by nucleic acid amplification tests is an attractive alternative to individual testing. As pooling can reduce the costs of testing as well as labor, it has been advocated for use in resource-poor settings. However, it has neither been widely adopted nor evaluated for use in developing countries. We evaluated the practical use of pooling first-catch urine (FCU) specimens for the detection of C. trachomatis and N. gonorrhoeae from 690 men in Mumbai, India, by PCR. FCU, urethral smears, and swabs were collected from men seen at two sexually transmitted infection (STI) clinics. All laboratory testing was done at the Lokmanya Tilak General Hospital. Gram stain smears and culture isolation for N. gonorrhoeae were performed. Each FCU was tested individually and in pools using the Roche Amplicor PCR for C. trachomatis and N. gonorrhoeae with an internal control for inhibition. Specimen pools consisted of aliquots from five consecutively processed FCUs combined into an amplification tube. An optical density reading of > or =0.20 indicated a pool for which subsequent testing of individual samples was required. Prevalence by PCR on single specimens was 2.2% (15/690) for C. trachomatis and 5.4% (37/690) for N. gonorrhoeae. Compared to individual FCU results, pooling for C. trachomatis and N. gonorrhoeae had an overall sensitivity of 96.1% (50/52). Specificity was 96.5% (83/86) in that three pools required single testing that failed to identify a positive specimen. Pooling missed two positive specimens, decreased the inhibition rate, and saved 50.3% of reagent costs. In this resource-limited setting, the use of pooling to detect C. trachomatis and N. gonorrhoeae by PCR proved to be a simple, accurate, and cost-effective procedure compared to individual testing.

Nested-PCR and a new ELISA-based NovaLisa test kit for malaria diagnosis in an endemic area of Thailand.

PubMed

Thongdee, Pimwan; Chaijaroenkul, Wanna; Kuesap, Jiraporn; Na-Bangchang, Kesara

2014-08-01

Microscopy is considered as the gold standard for malaria diagnosis although its wide application is limited by the requirement of highly experienced microscopists. PCR and serological tests provide efficient diagnostic performance and have been applied for malaria diagnosis and research. The aim of this study was to investigate the diagnostic performance of nested PCR and a recently developed an ELISA-based new rapid diagnosis test (RDT), NovaLisa test kit, for diagnosis of malaria infection, using microscopic method as the gold standard. The performance of nested-PCR as a malaria diagnostic tool is excellent with respect to its high accuracy, sensitivity, specificity, and ability to discriminate Plasmodium species. The sensitivity and specificity of nested-PCR compared with the microscopic method for detection of Plasmodium falciparum, Plasmodium vivax, and P. falciparum/P. vivax mixed infection were 71.4 vs 100%, 100 vs 98.7%, and 100 vs 95.0%, respectively. The sensitivity and specificity of the ELISA-based NovaLisa test kit compared with the microscopic method for detection of Plasmodium genus were 89.0 vs 91.6%, respectively. NovaLisa test kit provided comparable diagnostic performance. Its relatively low cost, simplicity, and rapidity enables large scale field application.

Molecular Analysis of Ammonia-Oxidizing Bacteria of the β Subdivision of the Class Proteobacteria in Compost and Composted Materials

PubMed Central

Kowalchuk, George A.; Naoumenko, Zinaida S.; Derikx, Piet J. L.; Felske, Andreas; Stephen, John R.; Arkhipchenko, Irina A.

1999-01-01

Although the practice of composting animal wastes for use as biofertilizers has increased in recent years, little is known about the microorganisms responsible for the nitrogen transformations which occur in compost and during the composting process. Ammonia is the principle available nitrogenous compound in composting material, and the conversion of this compound to nitrite in the environment by chemolithotrophic ammonia-oxidizing bacteria is an essential step in nitrogen cycling. Therefore, the distribution of ammonia-oxidizing members of the β subdivision of the class Proteobacteria in a variety of composting materials was assessed by amplifying 16S ribosomal DNA (rDNA) and 16S rRNA by PCR and reverse transcriptase PCR (RT-PCR), respectively. The PCR and RT-PCR products were separated by denaturing gradient gel electrophoresis (DGGE) and were identified by hybridization with a hierarchical set of oligonucleotide probes designed to detect ammonia oxidizer-like sequence clusters in the genera Nitrosospira and Nitrosomonas. Ammonia oxidizer-like 16S rDNA was detected in almost all of the materials tested, including industrial and experimental composts, manure, and commercial biofertilizers. A comparison of the DGGE and hybridization results after specific PCR and RT-PCR suggested that not all of the different ammonia oxidizer groups detected in compost are equally active. amoA, the gene encoding the active-site-containing subunit of ammonia monooxygenase, was also targeted by PCR, and template concentrations were estimated by competitive PCR. Detection of ammonia-oxidizing bacteria in the composts tested suggested that such materials may not be biologically inert with respect to nitrification and that the fate of nitrogen during composting and compost storage may be affected by the presence of these organisms. PMID:9925559

Evaluation of RIDA®GENE norovirus GI/GII real time RT-PCR using stool specimens collected from children and adults with acute gastroenteritis.

PubMed

Kanwar, N; Hassan, F; Barclay, L; Langley, C; Vinjé, J; Bryant, P W; George, K St; Mosher, L; Matthews-Greer, J M; Rocha, M A; Beenhouwer, D O; Harrison, C J; Moffatt, M; Shastri, N; Selvarangan, R

2018-04-10

Norovirus is the leading cause of epidemic and sporadic acute gastroenteritis (AGE) in the United States. Widespread prevalence necessitates implementation of accurate norovirus detection assays in clinical diagnostic laboratories. To evaluate RIDA ® GENE norovirus GI/GII real-time RT-PCR assay (RGN RT-PCR) using stool samples from patients with sporadic AGE. Patients between 14 days to 101 years of age with symptoms of AGE were enrolled prospectively at four sites across the United States during 2014-2015. Stool specimens were screened for the presence of norovirus RNA by the RGN RT-PCR assay. Results were compared with a reference method that included conventional RT-PCR and sequencing of a partial region of the 5'end of the norovirus ORF2 gene. A total of 259 (36.0%) of 719 specimens tested positive for norovirus by the reference method. The RGN RT-PCR assay detected norovirus in 244 (94%) of these 259 norovirus positive specimens. The sensitivity and specificity (95% confidence interval) of the RGN RT-PCR assay for detecting norovirus genogroup (G) I was 82.8% (63.5-93.5) and 99.1% (98.0-99.6) and for GII was 94.8% (90.8-97.2) and 98.6% (96.9-99.4), respectively. Seven specimens tested positive by the RGN-RT PCR that were negative by the reference method. The fifteen false negative samples were typed as GII.4 Sydney, GII.13, GI.3, GI.5, GI.2, GII.1, and GII.3 in the reference method. The RGN RT-PCR assay had a high sensitivity and specificity for the detection of norovirus in stool specimens from patients with sporadic AGE. Copyright © 2018. Published by Elsevier B.V.

The principle and application of new PCR Technologies

NASA Astrophysics Data System (ADS)

Yu, Miao; Cao, Yue; Ji, Yubin

2017-12-01

Polymerase chain reaction (PCR) is essentially a selective DNA amplification technique commonlyapplied for genetic testing and molecular diagnosis because of its high specificity and sensitivity.PCR technologies as the key of molecular biology, has realized that the qualitative detection of absolute quantitative has been changed. It has produced a variety of new PCR technologies, such as extreme PCR, photonic PCR, o-amplification at lower denaturation temperature PCR, nanoparticle PCR and so on. In this paper, the principle and application of PCR technologies are reviewed, and its development is prospected too.

Improved PCR-Based Detection of Soil Transmitted Helminth Infections Using a Next-Generation Sequencing Approach to Assay Design.

PubMed

Pilotte, Nils; Papaiakovou, Marina; Grant, Jessica R; Bierwert, Lou Ann; Llewellyn, Stacey; McCarthy, James S; Williams, Steven A

2016-03-01

The soil transmitted helminths are a group of parasitic worms responsible for extensive morbidity in many of the world's most economically depressed locations. With growing emphasis on disease mapping and eradication, the availability of accurate and cost-effective diagnostic measures is of paramount importance to global control and elimination efforts. While real-time PCR-based molecular detection assays have shown great promise, to date, these assays have utilized sub-optimal targets. By performing next-generation sequencing-based repeat analyses, we have identified high copy-number, non-coding DNA sequences from a series of soil transmitted pathogens. We have used these repetitive DNA elements as targets in the development of novel, multi-parallel, PCR-based diagnostic assays. Utilizing next-generation sequencing and the Galaxy-based RepeatExplorer web server, we performed repeat DNA analysis on five species of soil transmitted helminths (Necator americanus, Ancylostoma duodenale, Trichuris trichiura, Ascaris lumbricoides, and Strongyloides stercoralis). Employing high copy-number, non-coding repeat DNA sequences as targets, novel real-time PCR assays were designed, and assays were tested against established molecular detection methods. Each assay provided consistent detection of genomic DNA at quantities of 2 fg or less, demonstrated species-specificity, and showed an improved limit of detection over the existing, proven PCR-based assay. The utilization of next-generation sequencing-based repeat DNA analysis methodologies for the identification of molecular diagnostic targets has the ability to improve assay species-specificity and limits of detection. By exploiting such high copy-number repeat sequences, the assays described here will facilitate soil transmitted helminth diagnostic efforts. We recommend similar analyses when designing PCR-based diagnostic tests for the detection of other eukaryotic pathogens.

Detection methods for human enteric viruses in representative foods.

PubMed

Leggitt, P R; Jaykus, L A

2000-12-01

Although viral foodborne disease is a significant problem, foods are rarely tested for viral contamination, and when done, testing is limited to shellfish commodities. In this work, we report a method to extract and detect human enteric viruses from alternative food commodities using an elution-concentration approach followed by detection using reverse transcription-polymerase chain reaction (RT-PCR). Fifty-gram lettuce or hamburger samples were artificially inoculated with poliovirus type 1 (PV1), hepatitis A virus (HAV), or the Norwalk virus and processed by the sequential steps of homogenization, filtration, Freon extraction (hamburger), and polyethylene glycol (PEG) precipitation. To reduce volumes further and remove RT-PCR inhibitors, a secondary PEG precipitation was necessary, resulting in an overall 10- to 20-fold sample size reduction from 50 g to 3 to 5 ml. Virus recoveries in secondary PEG concentrates ranged from 10 to 70% for PV1 and 2 to 4% for HAV as evaluated by mammalian cell culture infectivity assay. Total RNA from PEG concentrates was extracted to a small volume (30 to 40 microl) and subjected to RT-PCR amplification of viral RNA sequences. Detection limit studies indicated that viral RNA was consistently detected by RT-PCR at initial inoculum levels > or =102 PFU/50-g food sample for PV1 and > or =10(3) PFU/50-g food sample for HAV. In similar studies with the Norwalk virus, detection at inoculum levels > or =1.5 X 10(3) PCR-amplifiable units/50-g sample for both food products was possible. All RT-PCR amplicons were confirmed by subsequent Southern hybridization. The procedure reported represents progress toward the development of methods to detect human enteric viral contamination in foods other than shellfish.

Development and accuracy of quantitative real-time polymerase chain reaction assays for detection and quantification of enterotoxigenic Escherichia coli (ETEC) heat labile and heat stable toxin genes in travelers' diarrhea samples.

PubMed

Youmans, Bonnie P; Ajami, Nadim J; Jiang, Zhi-Dong; Petrosino, Joseph F; DuPont, Herbert L; Highlander, Sarah K

2014-01-01

Enterotoxigenic Escherichia coli (ETEC), the leading bacterial pathogen of travelers' diarrhea, is routinely detected by an established DNA hybridization protocol that is neither sensitive nor quantitative. Quantitative real-time polymerase chain reaction (qPCR) assays that detect the ETEC toxin genes eltA, sta1, and sta2 in clinical stool samples were developed and tested using donor stool inoculated with known quantities of ETEC bacteria. The sensitivity of the qPCR assays is 89%, compared with 22% for the DNA hybridization assay, and the limits of detection are 10,000-fold lower than the DNA hybridization assays performed in parallel. Ninety-three clinical stool samples, previously characterized by DNA hybridization, were tested using the new ETEC qPCR assays. Discordant toxin profiles were observed for 22 samples, notably, four samples originally typed as ETEC negative were ETEC positive. The qPCR assays are unique in their sensitivity and ability to quantify the three toxin genes in clinical stool samples.

Detection of respiratory viruses on air filters from aircraft.

PubMed

Korves, T M; Johnson, D; Jones, B W; Watson, J; Wolk, D M; Hwang, G M

2011-09-01

To evaluate the feasibility of identifying viruses from aircraft cabin air, we evaluated whether respiratory viruses trapped by commercial aircraft air filters can be extracted and detected using a multiplex PCR, bead-based assay. The ResPlex II assay was first tested for its ability to detect inactivated viruses applied to new filter material; all 18 applications of virus at a high concentration were detected. The ResPlex II assay was then used to test for 18 respiratory viruses on 48 used air filter samples from commercial aircraft. Three samples tested positive for viruses, and three viruses were detected: rhinovirus, influenza A and influenza B. For 33 of 48 samples, internal PCR controls performed suboptimally, suggesting sample matrix effect. In some cases, influenza and rhinovirus RNA can be detected on aircraft air filters, even more than 10 days after the filters were removed from aircraft. With protocol modifications to overcome PCR inhibition, air filter sampling and the ResPlex II assay could be used to characterize viruses in aircraft cabin air. Information about viruses in aircraft could support public health measures to reduce disease transmission within aircraft and between cities. © The MITRE corporation. Letters in Applied Microbiology © 2011 The Society for Applied Microbiology.

Broad-range real-time PCR assay for the rapid identification of cell-line contaminants and clinically important mollicute species.

PubMed

Störmer, Melanie; Vollmer, Tanja; Henrich, Birgit; Kleesiek, Knut; Dreier, Jens

2009-04-01

Polymerase chain reaction assays have become widely used methods of confirming the presence of Mollicutes species in clinical samples and cell cultures. We have developed a broad-range real-time PCR assay using the locked nucleic acid technology to detect mollicute species causing human infection and cell line contamination. Primers and probes specifically for the conserved regions of the mycoplasmal tuf gene (encoding elongation factor Tu) were designed. Cell culture supernatants, clinical specimens (vaginal swabs, sputum, cryopreserved heart valve tissues), and reference strains were tested for mollicute contamination as well as to exclude cross-reaction to human nucleic acids and other bacterial species. Nucleic acids were extracted using magnetic separation technology. The coamplification of the human beta2-microglobulin DNA served as an internal control. The PCR assay was highly specific and obtained an analytical sensitivity of one copy per microl sample. The 95% detection limit was calculated to 10 copies per microl sample for Mycoplasma pneumoniae and M. orale. No false-positive results were observed due to cross-reaction of walled bacterial, fungal, and human nucleic acids. To evaluate the PCR, we compared the results to two commercialized test systems. Moreover, in combination with a previously developed broad-range RT-PCR assay for the detection of bacteria in blood products, both mollicute and walled bacterial contamination can be detected simultaneously using multiplex real-time RT-PCR.

A new real-time PCR protocol for detection of avian haemosporidians.

PubMed

Bell, Jeffrey A; Weckstein, Jason D; Fecchio, Alan; Tkach, Vasyl V

2015-07-19

Birds possess the most diverse assemblage of haemosporidian parasites; including three genera, Plasmodium, Haemoproteus, and Leucocytozoon. Currently there are over 200 morphologically identified avian haemosporidian species, although true species richness is unknown due to great genetic diversity and insufficient sampling in highly diverse regions. Studies aimed at surveying haemosporidian diversity involve collecting and screening samples from hundreds to thousands of individuals. Currently, screening relies on microscopy and/or single or nested standard PCR. Although effective, these methods are time and resource consuming, and in the case of microscopy require substantial expertise. Here we report a newly developed real-time PCR protocol designed to quickly and reliably detect all three genera of avian haemosporidians in a single biochemical reaction. Using available DNA sequences from avian haemosporidians we designed primers R330F and R480RL, which flank a 182 base pair fragment of mitochondrial conserved rDNA. These primers were initially tested using real-time PCR on samples from Malawi, Africa, previously screened for avian haemosporidians using traditional nested PCR. Our real time protocol was further tested on 94 samples from the Cerrado biome of Brazil, previously screened using a single PCR assay for haemosporidian parasites. These samples were also amplified using modified nested PCR protocols, allowing for comparisons between the three different screening methods (single PCR, nested PCR, real-time PCR). The real-time PCR protocol successfully identified all three genera of avian haemosporidians from both single and mixed infections previously detected from Malawi. There was no significant difference between the three different screening protocols used for the 94 samples from the Brazilian Cerrado (χ(2) = 0.3429, df = 2, P = 0.842). After proving effective, the real-time protocol was used to screen 2113 Brazilian samples, identifying 693 positive samples. Our real-time PCR assay proved as effective as two widely used molecular screening techniques, single PCR and nested PCR. However, the real-time protocol has the distinct advantage of detecting all three genera in a single reaction, which significantly increases efficiency by greatly decreasing screening time and cost. Our real-time PCR protocol is therefore a valuable tool in the quickly expanding field of avian haemosporidian research.

Rapid and accurate detection of KRAS mutations in colorectal cancers using the isothermal-based optical sensor for companion diagnostics

PubMed Central

Koo, Bonhan; Lee, Tae Yoon; Lee, Jeong Hoon; Shin, Yong; Lim, Seok-Byung

2017-01-01

Although KRAS mutational status testing is becoming a companion diagnostic tool for managing patients with colorectal cancer (CRC), there are still several difficulties when analyzing KRAS mutations using the existing assays, particularly with regard to low sensitivity, its time-consuming, and the need for large instruments. We developed a rapid, sensitive, and specific mutation detection assay based on the bio-photonic sensor termed ISAD (isothermal solid-phase amplification/detection), and used it to analyze KRAS gene mutations in human clinical samples. To validate the ISAD-KRAS assay for use in clinical diagnostics, we examined for hotspot KRAS mutations (codon 12 and codon 13) in 70 CRC specimens using PCR and direct sequencing methods. In a serial dilution study, ISAD-KRAS could detect mutations in a sample containing only 1% of the mutant allele in a mixture of wild-type DNA, whereas both PCR and direct sequencing methods could detect mutations in a sample containing approximately 30% of mutant cells. The results of the ISAD-KRAS assay from 70 clinical samples matched those from PCR and direct sequencing, except in 5 cases, wherein ISAD-KRAS could detect mutations that were not detected by PCR and direct sequencing. We also found that the sensitivity and specificity of ISAD-KRAS were 100% within 30 min. The ISAD-KRAS assay provides a rapid, highly sensitive, and label-free method for KRAS mutation testing, and can serve as a robust and near patient testing approach for the rapid detection of patients most likely to respond to anti-EGFR drugs. PMID:29137388

Sensitive and Rapid Detection of Viable Giardia Cysts and Cryptosporidium parvum Oocysts in Large-Volume Water Samples with Wound Fiberglass Cartridge Filters and Reverse Transcription-PCR

PubMed Central

Kaucner, Christine; Stinear, Timothy

1998-01-01

We recently described a reverse transcription-PCR (RT-PCR) for detecting low numbers of viable Cryptosporidium parvum oocysts spiked into clarified environmental water concentrates. We have now modified the assay for direct analysis of primary sample concentrates with simultaneous detection of viable C. parvum oocysts, Giardia cysts, and a novel type of internal positive control (IPC). The IPC was designed to assess both efficiency of mRNA isolation and potential RT-PCR inhibition. Sensitivity testing showed that low numbers of organisms, in the range of a single viable cyst and oocyst, could be detected when spiked into 100-μl packed pellet volumes of concentrates from creek and river water samples. The RT-PCR was compared with an immunofluorescence (IF) assay by analyzing 29 nonspiked environmental water samples. Sample volumes of 20 to 1,500 liters were concentrated with a wound fiberglass cartridge filter. Frequency of detection for viable Giardia cysts increased from 24% by IF microscopy to 69% by RT-PCR. Viable C. parvum oocysts were detected only once by RT-PCR (3%) in contrast to detection of viable Cryptosporidium spp. in four samples by IF microscopy (14%), suggesting that Cryptosporidium species other than C. parvum were present in the water. This combination of the large-volume sampling method with RT-PCR represents a significant advance in terms of protozoan pathogen monitoring and in the wider application of PCR technology to this field of microbiology. PMID:9572946

Integrative approach to diagnosis of genital human papillomaviruses (HPV) infection of female.

PubMed

Dunjic, Momir; Stanisic, Slavisa; Krstic, Dejan; Stanisic, Miodrag; Ignjatic, Z Jovanovic; Dunjic, Marija

2014-01-01

Human papillomavirus (HPV) is a virus from the papillomavirus family that is capable of infecting humans. Some types of HPVs cause warts, while others can lead to cancers of the cervix, vulva, vagina, penis, oropharynx and anus. High-risk human papillomavirus (hr HPV) has been detected in almost all cervical squamous cell carcinomas and adenocarcinomas. All patients examined by colposcopy. Cervical swab is routinely done and patients are screened with both HPV DNA by Real Time Polimerase Chain Reaction (RT PCR) testing and Pap testing. Pictures obtained by colposcopy were examined by indirect Bi-Digital O-Ring Test (BDORT) by using reference control substance (RCS): HPV 16, HPV 18, and Integrin α5 β1. BDORT was developed by Prof. Omura Y. of New York and received U.S. patent in 1993. For detection of HPV DNA we used RT PCR and standard Qiagen method which detect 18 types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 6, 11, 42, 43, 44) of HPV from smear. From 63 patients where is BDORT indicated presence of HPV, in 49 patients (77.8%) RT PCR confirmed presence of HPV. From 63 patients in 54 patients (85.7%), we detected, by colposcopic exam, some kind of lesions associated with HPV infection. Results obtained by RT PCR: one type (1/18) of DNA HPV in 25 patients (51.02%), 2 types (2/18) in 15 patients (30.61%) and 3 types (3/18) in 9 patients (18.37%). Although BDORT results usually have higher sensitivity and detection rate is much higher, it can be used together with RT PCR in detection of HPV and cervical lesions associated with HPV infection.

A multiplex PCR/LDR assay for simultaneous detection and identification of the NIAID category B bacterial food and water-borne pathogens.

PubMed

Rundell, Mark S; Pingle, Maneesh; Das, Sanchita; Hussain, Aashiq; Ocheretina, Oksana; Charles, Macarthur; Larone, Davise H; Spitzer, Eric D; Golightly, Linnie; Barany, Francis

2014-06-01

Enteric pathogens that cause gastroenteritis remain a major global health concern. The goal of this study was to develop a multiplex PCR/ligation detection reaction (LDR) assay for the detection of all NIAID category B bacterial food and water-borne pathogens directly from stool specimens. To validate the PCR/LDR assay, clinical isolates of Campylobacter spp., Vibrio spp., Shigella spp., Salmonella spp., Listeria monocytogenes, Yersinia enterocolitica, and diarrheagenic Escherichia coli were tested. The sensitivity and specificity of the assay were assessed using a large number of seeded culture-negative stool specimens and a smaller set of clinical specimens from Haiti. The overall sensitivity ranged from 91% to 100% (median 100%) depending on the species. For the majority of organisms, the sensitivity was 100%. The overall specificity based on initial testing ranged from 98% to 100% depending on the species. After additional testing of discordant samples, the lowest specificity was 99.4%. PCR/LDR detected additional category B agents (particularly diarrheagenic E. coli) in 11/40 specimens from Haiti that were culture-positive for V. cholerae and in approximately 1% of routine culture-negative stool specimens from a hospital in New York. This study demonstrated the ability of the PCR/LDR assay to detect a large comprehensive panel of category B enteric bacterial pathogens as well as mixed infections. This type of assay has the potential to provide earlier warnings of possible public health threats and more accurate surveillance of food and water-borne pathogens. Copyright © 2014 Elsevier Inc. All rights reserved.

Rapid identification of oral isolates of Aggregatibacter actinomycetemcomitans obtained from humans and primates by an ultrafast super convection based polymerase chain reaction

PubMed Central

Karched, M.; Furgang, D.; Sawalha, N.; Fine, D.H.

2017-01-01

Aggregatibacter actinomycetemcomitans is a Gram negative oral bacterium associated with localized aggressive periodontitis (LAP). Detection of A. actinomycetemcomitans in clinical samples is routinely done by PCR. Our aim was to develop a rapid and reliable PCR method that can be used as a chair-side tool to detect A. actinomycetemcomitans in clinical samples. Sensitivity and specificity assessment was performed on buccal and plaque samples obtained from 40 adolescents enrolled in an ongoing LAP study by comparing 20 A. actinomycetemcomitans-positive subjects and 20 who were negative. In a second study, A. actinomycetemcomitans presence was tested in oral samples from eighty-six primates that included rhesus monkeys, chimpanzees, marmosets, tamarins and baboons. All samples were processed for detection of A. actinomycetemcomitans by means of culture, conventional PCR (cPCR) and rapid PCR (rPCR) using a Super Convection based AmpXpress thermal cycler (AlphaHelix, Sweden). For human samples, culture, cPCR and rPCR showed perfect agreement. Using this method A. actinomycetemcomitans was detected in 27 of 32 rhesus monkeys, 4 of 8 chimpanzees and 1 of 34 marmosets. Rapidity of AmpXpress thermal cycler, combined with Ready-To-Go PCR beads (GE Life sciences), a quick DNA extraction kit (Epicentre Biotechnologies, Madison, Wisconsin, USA) and a bufferless fast agarose gel system, made it possible to obtain results on A. actinomycetemcomitans detection within 35 min. We conclude that AmpXpress fast PCR can be conveniently used as a chair-side tool for rapid detection of A. actinomycetemcomitans in clinical samples. PMID:22326236

Validation of PCR methods for quantitation of genetically modified plants in food.

PubMed

Hübner, P; Waiblinger, H U; Pietsch, K; Brodmann, P

2001-01-01

For enforcement of the recently introduced labeling threshold for genetically modified organisms (GMOs) in food ingredients, quantitative detection methods such as quantitative competitive (QC-PCR) and real-time PCR are applied by official food control laboratories. The experiences of 3 European food control laboratories in validating such methods were compared to describe realistic performance characteristics of quantitative PCR detection methods. The limit of quantitation (LOQ) of GMO-specific, real-time PCR was experimentally determined to reach 30-50 target molecules, which is close to theoretical prediction. Starting PCR with 200 ng genomic plant DNA, the LOQ depends primarily on the genome size of the target plant and ranges from 0.02% for rice to 0.7% for wheat. The precision of quantitative PCR detection methods, expressed as relative standard deviation (RSD), varied from 10 to 30%. Using Bt176 corn containing test samples and applying Bt176 specific QC-PCR, mean values deviated from true values by -7to 18%, with an average of 2+/-10%. Ruggedness of real-time PCR detection methods was assessed in an interlaboratory study analyzing commercial, homogeneous food samples. Roundup Ready soybean DNA contents were determined in the range of 0.3 to 36%, relative to soybean DNA, with RSDs of about 25%. Taking the precision of quantitative PCR detection methods into account, suitable sample plans and sample sizes for GMO analysis are suggested. Because quantitative GMO detection methods measure GMO contents of samples in relation to reference material (calibrants), high priority must be given to international agreements and standardization on certified reference materials.

Noninvasive screening for genital chlamydial infections in asymptomatic men: Strategies and costs using a urine PCR assay

PubMed Central

Peeling, Rosanna W; Toye, Baldwin; Jessamine, Peter; Gemmill, Ian

1998-01-01

OBJECTIVE: To evaluate cost saving strategies to screen for genital chlamydial infection in men using polymerase chain reaction (PCR) technology. METHODS: Men with no urethral symptoms presenting to a sexually transmitted disease (STD) clinic were recruited. Study participants underwent a questionnaire interview. Urethral swabs were taken to perform a smear for polymorphonuclear leucocytes (PMN) and for the detection of Chlamydia trachomatis by culture and PCR. First-catch urine was collected for a leukocyte esterase test (LET) and PCR. RESULTS: C trachomatis infection was detected in 36 of 463 (7.8%) men. LET and PMN were positive in 10 (28%) and 12 (33%) infected men, respectively. Risk factors for chlamydial infection were younger than age 25 years, LET-positive, PMN-positive and STD contact (P<0.001). The direct cost of genital chlamydial infection in men in Canada has been previously estimated at $381/case. Based on a sensitivity of 90% for urine PCR, the estimated direct cost of testing all participants to detect 32 cases was $453/case. Using risk factors recommended in the Canadian STD Guidelines (age younger than 25 years, new partner, STD contact or unprotected sex), the same number of cases would have been detected by testing only 384 men at $376/case. Using age younger than 25 years or STD contact as the screening criterion, 78% of those infected would have been detected at $259/case, and no new cases would have been detected by adding LET-positive or PMN-positive as risk factors. CONCLUSION: Targeted screening for chlamydial infection using urine PCR assay and risk factors recommended in the Canadian guidelines could substantially reduce the cost of screening at a STD clinic setting. LET and PMN smear did not appear to be useful indicators of chlamydial infection in this population. PMID:22346549

Rapid and Sensitive Detection of Bartonella bacilliformis in Experimentally Infected Sand Flies by Loop-Mediated Isothermal Amplification (LAMP) of the Pap31 Gene

PubMed Central

Angkasekwinai, Nasikarn; Atkins, Erin H.; Johnson, Richard N.; Grieco, John P.; Ching, Wei Mei; Chao, Chien Chung

2014-01-01

Background Carrion' disease, caused by Bartonella bacilliformis, remains truly neglected due to its focal geographical nature. A wide spectrum of clinical manifestations, including asymptomatic bacteremia, and lack of a sensitive diagnostic test can potentially lead to a spread of the disease into non-endemic regions where competent sand fly vectors may be present. A reliable test capable of detecting B. bacilliformis is urgently needed. Our objective is to develop a loop-mediated isothermal amplification (LAMP) assay targeting the pap31 gene to detect B. bacilliformis. Methods and Findings The sensitivity of the LAMP was evaluated in comparison to qPCR using plasmid DNA containing the target gene and genomic DNA in the absence and presence of human or sand fly DNA. The detection limit of LAMP was 1 to 10 copies/µL, depending on the sample metrics. No cross-reaction was observed when testing against a panel of various closely related bacteria. The utility of the LAMP was further compared to qPCR by the examination of 74 Lutzomyia longipalpis sand flies artificially fed on blood spiked with B. bacilliformis and harvested at days (D) 1, 3, 5, 7 and 9 post feeding. Only 86% of sand flies at D1 and 63% of flies at D3 were positive by qPCR. LAMP was able to detect B. bacilliformis in all those flies confirmed positive by qPCR. However, none of the flies after D3 were positive by either LAMP or qPCR. In addition to demonstrating the sensitivity of the LAMP assay, these results suggest that B. bacilliformis cannot propagate in artificially fed L. longipalpis. Conclusions The LAMP assay is as sensitive as qPCR for the detection of B. bacilliformis and could be useful to support diagnosis of patients in low-resource settings and also to identify B. bacilliformis in the sand fly vector. PMID:25522230

Effects of topical anaesthetics and fluorescein on the real‐time PCR used for the diagnosis of Herpesviruses and Acanthamoeba keratitis

PubMed Central

Goldschmidt, P; Rostane, H; Saint‐Jean, C; Batellier, L; Alouch, C; Zito, E; Bourcier, T; Laroche, L; Chaumeil, C

2006-01-01

Background The early microbiological diagnosis of corneal infections may prevent the condition from worsening. Aim To study the potential interferences of oxybuprocain and fluorescein solutions used by ophthalmologists on the performances of the real‐time polymerase chain reaction (PCR) carried out as routine test for diagnosis of keratitis. Methods Quantified suspensions of Herpes simplex virus (HSV1), Varicella zoster virus (VZV), Cytomegalovirus (CMV) and Acanthamoeba with and without oxybuprocain or fluorescein added before DNA extraction were tested by real‐time PCR. Results The capacities of the real‐time PCR to detect HSV, VZV, CMV and Acanthamoeba were reduced by oxybuprocain and fluorescein. Both products diluted to 1/16 reduced the PCR detection capacities for more than 2 logs (DNA copies/sample). Conclusions The simultaneous introduction of fluorescein or topical anaesthetics into the tubes containing the specimens to be tested by PCR may lead to false negative results. Because corneal specimens for microbiological diagnosis of keratitis are obtained after topical administration of anaesthetics and corneal staining with fluorescein, ophthalmologists should be aware to rinse the eye surface intensively with appropriate eye solutions to minimise the risks of misdiagnosis. PMID:16899529

Effects of topical anaesthetics and fluorescein on the real-time PCR used for the diagnosis of Herpesviruses and Acanthamoeba keratitis.

PubMed

Goldschmidt, P; Rostane, H; Saint-Jean, C; Batellier, L; Alouch, C; Zito, E; Bourcier, T; Laroche, L; Chaumeil, C

2006-11-01

The early microbiological diagnosis of corneal infections may prevent the condition from worsening. To study the potential interferences of oxybuprocain and fluorescein solutions used by ophthalmologists on the performances of the real-time polymerase chain reaction (PCR) carried out as routine test for diagnosis of keratitis. Quantified suspensions of Herpes simplex virus (HSV1), Varicella zoster virus (VZV), Cytomegalovirus (CMV) and Acanthamoeba with and without oxybuprocain or fluorescein added before DNA extraction were tested by real-time PCR. The capacities of the real-time PCR to detect HSV, VZV, CMV and Acanthamoeba were reduced by oxybuprocain and fluorescein. Both products diluted to 1/16 reduced the PCR detection capacities for more than 2 logs (DNA copies/sample). The simultaneous introduction of fluorescein or topical anaesthetics into the tubes containing the specimens to be tested by PCR may lead to false negative results. Because corneal specimens for microbiological diagnosis of keratitis are obtained after topical administration of anaesthetics and corneal staining with fluorescein, ophthalmologists should be aware to rinse the eye surface intensively with appropriate eye solutions to minimise the risks of misdiagnosis.

A multiplex real-time PCR assay, based on invA and pagC genes, for the detection and quantification of Salmonella enterica from cattle lymph nodes.

PubMed

Bai, Jianfa; Trinetta, Valentina; Shi, Xiaorong; Noll, Lance W; Magossi, Gabriela; Zheng, Wanglong; Porter, Elizabeth P; Cernicchiaro, Natalia; Renter, David G; Nagaraja, Tiruvoor G

2018-05-01

Cattle lymph nodes can harbor Salmonella and potentially contaminate beef products. We have developed and validated a new real-time PCR (qPCR) assay for the detection and quantification of Salmonella enterica in cattle lymph nodes. The assay targets both the invA and pagC genes, the most conserved molecular targets in Salmonella enterica. An 18S rRNA gene assay that amplifies from cattle and other animal species was also included as an internal control. Available DNA sequences for invA, pagC and 18S rRNA genes were used for primer and probe selections. Three Salmonella serotypes, S. Typhimurium, S. Anatum, and S. Montevideo, were used to assess the assay's analytical sensitivity. Correlation coefficients of standard curves generated for each target and for all three serotypes were >99% and qPCR amplification efficiencies were between 93% and 110%. Assay sensitivity was also determined using standard curve data generated from Salmonella-negative cattle lymph nodes spiked with 10-fold dilutions of the three Salmonella serotypes. Assay specificity was determined using Salmonella culture method, and qPCR testing on 36 Salmonella strains representing 33 serotypes, 38 Salmonella strains of unknown serotypes, 252 E. coli strains representing 40 serogroups, and 31 other bacterial strains representing 18 different species. A collection of 647 cattle lymph node samples from steers procured from the Midwest region of the US were tested by the qPCR, and compared to culture-method of detection. Salmonella prevalence by qPCR for pre-enriched and enriched lymph nodes was 19.8% (128/647) and 94.9% (614/647), respectively. A majority of qPCR positive pre-enriched samples (105/128) were at concentrations between 10 4 and 10 5  CFU/mL. Culture method detected Salmonella in 7.7% (50/647) and 80.7% (522/647) of pre- and post-enriched samples, respectively; 96.0% (48/50) of pre-enriched and 99.4% (519/522) of post-enriched culture-positive samples were also positive by qPCR. More samples tested positive by qPCR than by culture method, indicating that the real-time PCR assay was more sensitive. Our data indicate that this triplex qPCR can be used to accurately detect and quantify Salmonella enterica strains from cattle lymph node samples. The assay may serve as a useful tool to monitor the prevalence of Salmonella in beef production systems. Copyright © 2018 Elsevier B.V. All rights reserved.

Rapid detection and differentiation of avian infectious bronchitis virus: an application of Mass genotype by melting temperature analysis in RT-qPCR using SYBR Green I

PubMed Central

OKINO, Cintia Hiromi; MONTASSIER, Maria de Fátima Silva; de OLIVEIRA, Andressa Peres; MONTASSIER, Helio José

2018-01-01

A method based on Melting Temperature analysis of Hypervariable regions (HVR) of S1 gene within a RT-qPCR was developed to detect different genotypes of avian infectious bronchitis virus (IBV) and identify the Mass genotype. The method was able to rapidly identify the Mass genotype among IBV field isolates, vaccine attenuated strains and reference M41 strain in allantoic liquid and also directly in tissues. The RT-qPCR developed detected the virus in both tracheal and pulmonary samples from M41-infected or H120-infected birds, in a larger post-infection period compared to detection by standard method of virus isolation. RT-qPCR method tested provided a sensitivity and rapid approach for screening on IBV detection and Mass genotyping from IBV isolates. PMID:29491226

Zika virus RNA polymerase chain reaction on the utility channel of a commercial nucleic acid testing system.

PubMed

Boujnan, Mohamed; Duits, Ashley J; Koppelman, Marco H G M

2018-03-01

Several countries have implemented safety strategies to reduce the risk of Zika virus (ZIKV) transmission through blood transfusion. These strategies have included nucleic acid amplification testing (NAT) of blood donations. In this study, a new real-time polymerase chain reaction (PCR) assay including internal control for the detection of ZIKV on the cobas omni Utility Channel (UC) on the cobas 6800 system is presented. PCR conditions and primer/probe concentrations were optimized on the LightCycler 480 instrument. Optimized conditions were transferred to the cobas omni UC on the cobas 6800 system. Subsequently, the limit of detection (LOD) in plasma and urine, genotype inclusivity, specificity, cross-reactivity, and clinical sensitivity were determined. The 95% LOD of the ZIKV PCR assay on the cobas 6800 system was 23.0 IU/mL (95% confidence interval [CI], 16.5-37.5) in plasma and 24.5 IU/mL (95% CI, 13.4-92.9) in urine. The assay detected African and Asian lineages of ZIKV. The specificity was 100%. The clinical concordance between the newly developed ZIKV PCR assay and the investigational Roche cobas Zika NAT test was 83% (24/29). We developed a sensitive ZIKV PCR assay on the cobas omni UC on the cobas 6800 system. The assay can be used for large-scale screening of blood donations for ZIKV or for testing of blood donors returning from areas with ZIKV to avoid temporal deferral. This study also demonstrates that the cobas omni UC on the cobas 6800 system can be used for in-house-developed PCR assays. © 2018 AABB.

Efficiency of Nested-PCR in Detecting Asymptomatic Cases toward Malaria Elimination Program in an Endemic Area of Iran.

PubMed

Turki, Habibollah; Raeisi, Ahmad; Malekzadeh, Kianoosh; Ghanbarnejad, Amin; Zoghi, Samaneh; Yeryan, Masoud; Abedi Nejad, Masoumeh; Mohseni, Fatemeh; Shekari, Mohammad

2015-01-01

The aim of this study was to detect low parasite and asymptomatic malaria infections by means of three malaria diagnostic tests, in a low transmission region of Minab district, Hormozgan Province, southern Iran. Blood samples of 200 healthy volunteers from Bagh-e-Malek area were evaluated using microscopic, rapid diagnostic tests (RDT) and nested-PCR to inspect malaria parasite. The results showed no Plasmodium parasite in subjects by means of microscopy and RDT. However, 3 P. vivax positive samples (1.5%) were discovered by Nested-PCR while microscopy and RDT missed the cases. Microscopy as the gold standard method and RDT correctly identified 98.5% of cases, and molecular analysis is sensitive and reliable, especially in the detection of "asymptomatic" infections for active case surveillance. Regarding the existence of asymptomatic malaria in endemic area of Hormozgan, Iran, nested-PCR could be considered as a sensitive tool to interrupt malaria transmission in the country, beside the microscopic and RDT methods.

[Evaluation of the usefulness of various PCR method variations and nucleic acid hybridization for CMV infection in immunosuppressed patients].

PubMed

Siennicka, J; Trzcińska, A; Litwińska, B; Durlik, M; Seferyńska, I; Pałynyczko, G; Kańtoch, M

2000-01-01

In diagnosis of CMV infection various laboratory methods are used. The methods based on detection of viral nucleic acids have been introduced routinely in many laboratories. The aim of this study was to compare nucleic acid hybridisation method and various variants of PCR methods with respect to their ability to detect CMV DNA. The studied material comprised 60 blood samples from 19 patients including 13 renal transplant recipients and 6 with acute leukaemia. The samples were subjected to hybridisation (Murex Hybrid Capture System CMV DNA) and PCR carried out in 3 variants: with one pair of primers (single PCR), nested PCR and Digene SHARP System with detection of PCR product using a genetic probe in ELISA system. The sensitivity of the variants ranged from 10(0) particles of viral DNA in nested PCR to 10(2) in single PCR. The producer claimed the sensitivity of the hybridisation test to be 3 x 10(5) and it seems to be sufficient for detection of CMV infection. The obtained results show that sensitivity of hybridisation was comparable to that of single PCR and the possibility of obtaining quantitative results makes it superior, on efficacy of antiviral therapy, especially in monitoring CMV infection in immunossuppressed patients and in following the efficacy of antiviral treatment.

A novel mechanism for direct real-time polymerase chain reaction that does not require DNA isolation from prokaryotic cells.

PubMed

Soejima, Takashi; Xiao, Jin-Zhong; Abe, Fumiaki

2016-06-23

Typically, polymerase chain reaction (PCR) is performed after DNA isolation. Real-time PCR (qPCR), also known as direct qPCR in mammalian cells with weak membranes, is a common technique using crude samples subjected to preliminary boiling to elute DNA. However, applying this methodology to prokaryotic cells, which have solid cell walls, in contrast to mammalian cells which immediately burst in water, can result in poor detection. We successfully achieved PCR elongation with the addition of 1.3 cfu of Cronobacter muytjensii to a newly developed direct qPCR master mix without performing any crude DNA extraction (detection limit of 1.6 × 10(0) cfu/ml for the test sample compared with a detection limit of 1.6 × 10(3) cfu/ml primarily for crude (boiling) or classical DNA isolation). We revealed that the chromosomal DNA retained in prokaryotic cells can function as a PCR template, similarly to the mechanism in in situ PCR. Elucidating this reaction mechanism may contribute to the development of an innovative master mix for direct qPCR to detect genes in a single bacterium with solid cell walls and might lead to numerous novel findings in prokaryotic genomics research.

High performance of a new PCR-based urine assay for HPV-DNA detection and genotyping.

PubMed

Tanzi, Elisabetta; Bianchi, Silvia; Fasolo, Maria Michela; Frati, Elena R; Mazza, Francesca; Martinelli, Marianna; Colzani, Daniela; Beretta, Rosangela; Zappa, Alessandra; Orlando, Giovanna

2013-01-01

Human papillomavirus (HPV) testing has been proposed as a means of replacing or supporting conventional cervical screening (Pap test). However, both methods require the collection of cervical samples. Urine sample is easier and more acceptable to collect and could be helpful in facilitating cervical cancer screening. The aim of this study was to evaluate the sensitivity and specificity of urine testing compared to conventional cervical smear testing using a PCR-based method with a new, designed specifically primer set. Paired cervical and first voided urine samples collected from 107 women infected with HIV were subjected to HPV-DNA detection and genotyping using a PCR-based assay and a restriction fragment length polymorphism method. Sensitivity, specificity, Positive Predictive Value (PPV), and Negative Predictive Value (NPV) were calculated using the McNemar's test for differences. Concordance between tests was assessed using the Cohen's unweighted Kappa (k). HPV DNA was detected in 64.5% (95% CI: 55.1-73.1%) of both cytobrush and urine samples. High concordance rates of HPV-DNA detection (k = 0.96; 95% CI: 0.90-1.0) and of high risk-clade and low-risk genotyping in paired samples (k = 0.80; 95% CI: 0.67-0.92 and k = 0.74; 95% CI: 0.60-0.88, respectively) were observed. HPV-DNA detection in urine versus cervix testing revealed a sensitivity of 98.6% (95% CI: 93.1-99.9%) and a specificity of 97.4% (95% CI: 87.7-99.9%), with a very high NPV (97.4%; 95% CI: 87.7-99.9%). The PCR-based assay utilized in this study proved highly sensitive and specific for HPV-DNA detection and genotyping in urine samples. These data suggest that a urine-based assay would be a suitable and effective tool for epidemiological surveillance and, most of all, screening programs. Copyright © 2012 Wiley Periodicals, Inc.

EGFR mutation detection in ctDNA from NSCLC patient plasma: A cross-platform comparison of leading technologies to support the clinical development of AZD9291.

PubMed

Thress, Kenneth S; Brant, Roz; Carr, T Hedley; Dearden, Simon; Jenkins, Suzanne; Brown, Helen; Hammett, Tracey; Cantarini, Mireille; Barrett, J Carl

2015-12-01

To assess the ability of different technology platforms to detect epidermal growth factor receptor (EGFR) mutations, including T790M, from circulating tumor DNA (ctDNA) in advanced non-small cell lung cancer (NSCLC) patients. A comparison of multiple platforms for detecting EGFR mutations in plasma ctDNA was undertaken. Plasma samples were collected from patients entering the ongoing AURA trial (NCT01802632), investigating the safety, tolerability, and efficacy of AZD9291 in patients with EGFR-sensitizing mutation-positive NSCLC. Plasma was collected prior to AZD9291 dosing but following clinical progression on a previous EGFR-tyrosine kinase inhibitor (TKI). Extracted ctDNA was analyzed using two non-digital platforms (cobas(®) EGFR Mutation Test and therascreen™ EGFR amplification refractory mutation system assay) and two digital platforms (Droplet Digital™ PCR and BEAMing digital PCR [dPCR]). Preliminary assessment (38 samples) was conducted using all four platforms. For EGFR-TKI-sensitizing mutations, high sensitivity (78-100%) and specificity (93-100%) were observed using tissue as a non-reference standard. For the T790M mutation, the digital platforms outperformed the non-digital platforms. Subsequent assessment using 72 additional baseline plasma samples was conducted using the cobas(®) EGFR Mutation Test and BEAMing dPCR. The two platforms demonstrated high sensitivity (82-87%) and specificity (97%) for EGFR-sensitizing mutations. For the T790M mutation, the sensitivity and specificity were 73% and 67%, respectively, with the cobas(®) EGFR Mutation Test, and 81% and 58%, respectively, with BEAMing dPCR. Concordance between the platforms was >90%, showing that multiple platforms are capable of sensitive and specific detection of EGFR-TKI-sensitizing mutations from NSCLC patient plasma. The cobas(®) EGFR Mutation Test and BEAMing dPCR demonstrate a high sensitivity for T790M mutation detection. Genomic heterogeneity of T790M-mediated resistance may explain the reduced specificity observed with plasma-based detection of T790M mutations versus tissue. These data support the use of both platforms in the AZD9291 clinical development program. Copyright © 2015 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.

Use of polymerase chain reaction for the detection of Chlamydia trachomatis in ocular and nasopharyngeal specimens from infants with conjunctivitis.

PubMed

Hammerschlag, M R; Roblin, P M; Gelling, M; Tsumura, N; Jule, J E; Kutlin, A

1997-03-01

Chlamydia trachomatis is the most common identifiable infectious cause of neonatal conjunctivitis. Nonculture tests including enzyme immunoassays and direct fluorescent antibody tests have been shown to perform well for the diagnosis of chlamydial conjunctivitis with sensitivities and specificities > or = 90%. However, the performance with respiratory specimens has been less than satisfactory. We compared a new, commercially available polymerase chain reaction (PCR) assay, Roche AMPLICOR (Roche Diagnostic Systems, Branchburg, NJ) with culture for the detection of C. trachomatis in conjunctival and nasopharyngeal specimens from infants with conjunctivitis. We also evaluated AMPLICOR for the detection of C. trachomatis in the urine of mothers of positive infants. Ocular and nasopharyngeal specimens from 75 infants with conjunctivitis were obtained for culture and PCR. AMPLICOR was equivalent to culture for eye specimens and more sensitive than culture for nasopharyngeal specimens. The sensitivity, specificity and positive and negative predictive values of PCR compared with culture for conjunctival specimens were 92.3, 100, 100 and 98.4%, respectively. The sensitivity, specificity and positive and negative predictive values for nasopharyngeal specimens were 100, 97.2, 60 and 100%, respectively. We also detected C. trachomatis by PCR in the urine of 12 mothers of culture positive infants. PCR performed comparably to culture for detection of C. trachomatis in conjunctival and nasopharyngeal specimens from infants with conjunctivitis.

Lyme Borreliosis--the Utility of Improved Real-Time PCR Assay in the Detection of Borrelia burgdorferi Infections.

PubMed

Bil-Lula, Iwona; Matuszek, Patryk; Pfeiffer, Thomas; Woźniak, Mieczysław

2015-01-01

Infections of Borrelia burgdorferi sensu lato reveal clinical manifestations affecting numerous organs and tissues. The standard diagnostic procedure of these infections is quite simple if a positive history of tick exposure or typical erythema migrans appears. Lack of unequivocal clinical symptoms creates the necessity for further evaluation with laboratory tests. This study discusses the utility of a novel, improved, well-optimized, sensitive and highly specific quantitative real-time PCR assay for the diagnostics of infections caused by Borrelia burgdorferi sensu lato. We designed an improved, specific, highly sensitive real-time quantitative polymerase chain reaction (RQ-PCR) assay for the detection and quantification of all Borrelia burgdorferi genotypes. A wide validation effort was undertaken to ensure confidence in the highly sensitive and specific detection of B. burgdorferi. Due to high sensitivity and great specificity, as low as 1.6×10² copies of Borrelia per mL of whole blood could be detected. As much as 12 (3%) negative ELISA IgM results, 14 (2.8%) negative results of Line blot IgM, 11 (3.1%) and 7 (2.7%) of negative ELISA IgG and Line blot IgG results, respectively, were positive in real-time PCR. The data in this study confirms the high positive predictive value of real-time PCR test in the detection of Borrelia infections.

ELISA versus PCR for diagnosis of chronic Chagas disease: systematic review and meta-analysis

PubMed Central

2010-01-01

Background Most current guidelines recommend two serological tests to diagnose chronic Chagas disease. When serological tests are persistently inconclusive, some guidelines recommend molecular tests. The aim of this investigation was to review chronic Chagas disease diagnosis literature and to summarize results of ELISA and PCR performance. Methods A systematic review was conducted searching remote databases (MEDLINE, LILACS, EMBASE, SCOPUS and ISIWeb) and full texts bibliography for relevant abstracts. In addition, manufacturers of commercial tests were contacted. Original investigations were eligible if they estimated sensitivity and specificity, or reliability -or if their calculation was possible - of ELISA or PCR tests, for chronic Chagas disease. Results Heterogeneity was high within each test (ELISA and PCR) and threshold effect was detected only in a particular subgroup. Reference standard blinding partially explained heterogeneity in ELISA studies, and pooled sensitivity and specificity were 97.7% [96.7%-98.5%] and 96.3% [94.6%-97.6%] respectively. Commercial ELISA with recombinant antigens studied in phase three investigations partially explained heterogeneity, and pooled sensitivity and specificity were 99.3% [97.9%-99.9%] and 97.5% [88.5%-99.5%] respectively. ELISA's reliability was seldom studied but was considered acceptable. PCR heterogeneity was not explained, but a threshold effect was detected in three groups created by using guanidine and boiling the sample before DNA extraction. PCR sensitivity is likely to be between 50% and 90%, while its specificity is close to 100%. PCR reliability was never studied. Conclusions Both conventional and recombinant based ELISA give useful information, however there are commercial tests without technical reports and therefore were not included in this review. Physicians need to have access to technical reports to understand if these serological tests are similar to those included in this review and therefore correctly order and interpret test results. Currently, PCR should not be used in clinical practice for chronic Chagas disease diagnosis and there is no PCR test commercially available for this purpose. Tests limitations and directions for future research are discussed. PMID:21108793

Identification of Histoplasma capsulatum from culture extracts by real-time PCR.

PubMed

Martagon-Villamil, Jose; Shrestha, Nabin; Sholtis, Mary; Isada, Carlos M; Hall, Gerri S; Bryne, Terry; Lodge, Barbara A; Reller, L Barth; Procop, Gary W

2003-03-01

We designed and tested a real-time LightCycler PCR assay for Histoplasma capsulatum that correctly identified the 34 H. capsulatum isolates in a battery of 107 fungal isolates tested and also detected H. capsulatum in clinical specimens from three patients that were culture positive for this organism.

Diagnostic sensitivity and specificity of in situ hybridization and immunohistochemistry for Eastern equine encephalitis virus and West Nile virus in formalin-fixed, paraffin-embedded brain tissue of horses.

PubMed

Pennick, Kate E; McKnight, Christy A; Patterson, Jon S; Latimer, Kenneth S; Maes, Roger K; Wise, Annabel G; Kiupel, Matti

2012-03-01

Immunohistochemistry (IHC) and in situ hybridization (ISH) can be used either to detect or to differentiate between Eastern equine encephalitis virus (EEEV) and West Nile virus (WNV) within formalin-fixed, paraffin-embedded (FFPE) brain tissue of horses. To compare the diagnostic sensitivity and specificity of ISH and IHC, FFPE brain tissue from 20 EEEV-positive horses and 16 WNV-positive horses were tested with both EEEV and WNV oligoprobes and EEEV- and WNV-specific antibodies. Reverse transcription polymerase chain reaction (RT-PCR) for detection of EEEV and WNV was used as the gold standard to confirm infection. All horses that tested positive for EEEV by RT-PCR also tested positive by IHC and ISH, except for 1 case that was false-negative by ISH. In contrast, all horses that tested positive for WNV by RT-PCR tested negative by IHC and only 2 horses tested positive by ISH. No false-positives were detected with either method for both viruses. Both IHC and ISH are highly specific and sensitive diagnostic methods to detect EEEV in equine FFPE brain tissues, although neither appear effective for the diagnosis of WNV in equine neurologic cases.

Multiplex Real-Time PCR for Detection of Staphylococcus aureus, mecA and Panton-Valentine Leukocidin (PVL) Genes from Selective Enrichments from Animals and Retail Meat

PubMed Central

Velasco, Valeria; Sherwood, Julie S.; Rojas-García, Pedro P.; Logue, Catherine M.

2014-01-01

The aim of this study was to compare a real-time PCR assay, with a conventional culture/PCR method, to detect S. aureus, mecA and Panton-Valentine Leukocidin (PVL) genes in animals and retail meat, using a two-step selective enrichment protocol. A total of 234 samples were examined (77 animal nasal swabs, 112 retail raw meat, and 45 deli meat). The multiplex real-time PCR targeted the genes: nuc (identification of S. aureus), mecA (associated with methicillin resistance) and PVL (virulence factor), and the primary and secondary enrichment samples were assessed. The conventional culture/PCR method included the two-step selective enrichment, selective plating, biochemical testing, and multiplex PCR for confirmation. The conventional culture/PCR method recovered 95/234 positive S. aureus samples. Application of real-time PCR on samples following primary and secondary enrichment detected S. aureus in 111/234 and 120/234 samples respectively. For detection of S. aureus, the kappa statistic was 0.68–0.88 (from substantial to almost perfect agreement) and 0.29–0.77 (from fair to substantial agreement) for primary and secondary enrichments, using real-time PCR. For detection of mecA gene, the kappa statistic was 0–0.49 (from no agreement beyond that expected by chance to moderate agreement) for primary and secondary enrichment samples. Two pork samples were mecA gene positive by all methods. The real-time PCR assay detected the mecA gene in samples that were negative for S. aureus, but positive for Staphylococcus spp. The PVL gene was not detected in any sample by the conventional culture/PCR method or the real-time PCR assay. Among S. aureus isolated by conventional culture/PCR method, the sequence type ST398, and multi-drug resistant strains were found in animals and raw meat samples. The real-time PCR assay may be recommended as a rapid method for detection of S. aureus and the mecA gene, with further confirmation of methicillin-resistant S. aureus (MRSA) using the standard culture method. PMID:24849624

Multiplex real-time PCR for detection of Staphylococcus aureus, mecA and Panton-Valentine Leukocidin (PVL) genes from selective enrichments from animals and retail meat.

PubMed

Velasco, Valeria; Sherwood, Julie S; Rojas-García, Pedro P; Logue, Catherine M

2014-01-01

The aim of this study was to compare a real-time PCR assay, with a conventional culture/PCR method, to detect S. aureus, mecA and Panton-Valentine Leukocidin (PVL) genes in animals and retail meat, using a two-step selective enrichment protocol. A total of 234 samples were examined (77 animal nasal swabs, 112 retail raw meat, and 45 deli meat). The multiplex real-time PCR targeted the genes: nuc (identification of S. aureus), mecA (associated with methicillin resistance) and PVL (virulence factor), and the primary and secondary enrichment samples were assessed. The conventional culture/PCR method included the two-step selective enrichment, selective plating, biochemical testing, and multiplex PCR for confirmation. The conventional culture/PCR method recovered 95/234 positive S. aureus samples. Application of real-time PCR on samples following primary and secondary enrichment detected S. aureus in 111/234 and 120/234 samples respectively. For detection of S. aureus, the kappa statistic was 0.68-0.88 (from substantial to almost perfect agreement) and 0.29-0.77 (from fair to substantial agreement) for primary and secondary enrichments, using real-time PCR. For detection of mecA gene, the kappa statistic was 0-0.49 (from no agreement beyond that expected by chance to moderate agreement) for primary and secondary enrichment samples. Two pork samples were mecA gene positive by all methods. The real-time PCR assay detected the mecA gene in samples that were negative for S. aureus, but positive for Staphylococcus spp. The PVL gene was not detected in any sample by the conventional culture/PCR method or the real-time PCR assay. Among S. aureus isolated by conventional culture/PCR method, the sequence type ST398, and multi-drug resistant strains were found in animals and raw meat samples. The real-time PCR assay may be recommended as a rapid method for detection of S. aureus and the mecA gene, with further confirmation of methicillin-resistant S. aureus (MRSA) using the standard culture method.

Agreement Rate of Rapid Urease Test, Conventional PCR, and Scorpion Real-Time PCR in Detecting Helicobacter Pylori from Tonsillar Samples of Patients with Chronic Tonsillitis

PubMed Central

Najafipour, Reza; Farivar, Taghi Naserpour; Pahlevan, Ali Akbar; Johari, Pouran; Safdarian, Farshid; Asefzadeh, Mina

2012-01-01

Background: Helicobacter pylori is capable of inducing systemic inflammatory reactions through immunological processes. There are several methods to identify the presence of H. pylori in clinical samples including rapid urease test (RUT), conventional polymerase chain reaction (PCR), and the Scorpion real-time PCR. Aim: The aim of the present study is to compare the agreement rate of these tests in identifying H. pylori in tonsillar biopsy specimens collected from patients with chronic tonsillitis. Materials and Methods: A total of 103 tonsil biopsy samples from patients with clinical signs of chronic tonsillitis were examined with RUT, PCR, and Scorpion real-time PCR. The degree of agreement between the three tests was later calculated. Results: There was a poor degree of agreement between RUT and PCR and also RUT and Scorpion real-time PCR (Kappa=0.269 and 0.249, respectively). In contrast with RUT, there was a strong degree of agreement between PCR and Scorpion real-time PCR (Kappa=0.970). Conclusion: The presence of a strong agreement between the Scorpion real-time PCR and PCR as well as its technical advantage over the conventional PCR assay, made the Scorpion real-time PCR an appropriate laboratory test to investigate the presence of H. pylori in tonsillar biopsy specimens in patients suffering from chronic tonsillitis. PMID:22754245

Setting up a probe based, closed tube real-time PCR assay for focused detection of variable sequence alterations.

PubMed

Becságh, Péter; Szakács, Orsolya

2014-10-01

During diagnostic workflow when detecting sequence alterations, sometimes it is important to design an algorithm that includes screening and direct tests in combination. Normally the use of direct test, which is mainly sequencing, is limited. There is an increased need for effective screening tests, with "closed tube" during the whole process and therefore decreasing the risk of PCR product contamination. The aim of this study was to design such a closed tube, detection probe based screening assay to detect different kind of sequence alterations in the exon 11 of the human c-kit gene region. Inside this region there are variable possible deletions and single nucleotide changes. During assay setup, more probe chemistry formats were screened and tested. After some optimization steps the taqman probe format was selected.

Evaluation of different methods to detect methicillin resistance in Staphylococcus aureus (MRSA).

PubMed

Alipour, Farzad; Ahmadi, Malahat; Javadi, Shahram

2014-01-01

The studies suggest that dogs living with human are potential risk of becoming MRSA carrier and increased risk of infections caused by MRSA. Phenotypic methods to detect methicillin resistance in Staphylococcus aureus (MRSA) are inadequate. The objective of the present study was to determine methicillin resistance in S. aureus by phenotypic susceptibility test (oxacillin disk diffusion, cefoxitin disk diffusion, oxacillin screen agar) and molecular methods (PCR as a gold standard) and the latex agglutination test for the detection of PBP2a and to evaluate the results of these tests for its sensitivity and specificity. A total of 100 swab samples were taken from muzzle site, in more contact with human, of dogs and MRSA were isolated. Oxacillin (1 μg), cefoxitin (30 μg) disk diffusion and oxacillin screen agar method were used. The isolates were also subjected to latex agglutination test for detection of PBP2a and PCR to detect mecA gene. By PCR 37% of isolates show the presence of mecA. Latex agglutination was found to be the most sensitive (97.29%) and cefoxitin disk diffusion to be the most specific (96.82%) tests for detection of MRSA. Our finding showed that combining oxacillin screen agar or cefoxitin disk diffusion with latex agglutination improves sensitivity and specificity to detect methicillin resistance S. aureus (MRSA) isolates. Copyright © 2014 King Saud Bin Abdulaziz University for Health Sciences. Published by Elsevier Ltd. All rights reserved.

Real-time PCR assays for detection and quantification of aflatoxin-producing molds in foods.

PubMed

Rodríguez, Alicia; Rodríguez, Mar; Luque, M Isabel; Martín, Alberto; Córdoba, Juan J

2012-08-01

Aflatoxins are among the most toxic mycotoxins. Early detection and quantification of aflatoxin-producing species is crucial to improve food safety. In the present work, two protocols of real-time PCR (qPCR) based on SYBR Green and TaqMan were developed, and their sensitivity and specificity were evaluated. Primers and probes were designed from the o-methyltransferase gene (omt-1) involved in aflatoxin biosynthesis. Fifty-three mold strains representing aflatoxin producers and non-producers of different species, usually reported in food products, were used as references. All strains were tested for aflatoxins production by high-performance liquid chromatography-mass spectrometry (HPLC-MS). The functionality of the proposed qPCR method was demonstrated by the strong linear relationship of the standard curves constructed with the omt-1 gene copy number and Ct values for the different aflatoxin producers tested. The ability of the qPCR protocols to quantify aflatoxin-producing molds was evaluated in different artificially inoculated foods. A good linear correlation was obtained over the range 4 to 1 log cfu/g per reaction for all qPCR assays in the different food matrices (peanuts, spices and dry-fermented sausages). The detection limit in all inoculated foods ranged from 1 to 2 log cfu/g for SYBR Green and TaqMan assays. No significant effect was observed due to the different equipment, operator, and qPCR methodology used in the tests of repeatability and reproducibility for different foods. The proposed methods quantified with high efficiency the fungal load in foods. These qPCR protocols are proposed for use to quantify aflatoxin-producing molds in food products. Copyright © 2012 Elsevier Ltd. All rights reserved.

Detection of a substantial number of sub-microscopic Plasmodium falciparum infections by polymerase chain reaction: a potential threat to malaria control and diagnosis in Ethiopia

PubMed Central

2013-01-01

Background Prompt and effective malaria diagnosis not only alleviates individual suffering, but also decreases malaria transmission at the community level. The commonly used diagnostic methods, microscopy and rapid diagnostic tests, are usually insensitive at very low-density parasitaemia. Molecular techniques, on the other hand, allow the detection of low-level, sub-microscopic parasitaemia. This study aimed to explore the presence of sub-microscopic Plasmodium falciparum infections using polymerase chain reaction (PCR). The PCR-based parasite prevalence was compared against microscopy and rapid diagnostic test (RDT). Methods This study used 1,453 blood samples collected from clinical patients and sub-clinical subjects to determine the prevalence of sub-microscopic P. falciparum carriages. Subsets of RDT and microscopy negative blood samples were tested by PCR while all RDT and microscopically confirmed P. falciparum-infected samples were subjected to PCR. Finger-prick blood samples spotted on filter paper were used for parasite genomic DNA extraction. Results The prevalence of sub-microscopic P. falciparum carriage was 19.2% (77/400) (95% CI = 15. 4–23.1). Microscopy-based prevalence of P. falciparum infection was 3.7% (54/1,453) while the prevalence was 6.9% (100/1,453) using RDT alone. Using microscopy and PCR, the estimated parasite prevalence was 20.6% if PCR were performed in 1,453 blood samples. The prevalence was estimated to be 22.7% if RDT and PCR were used. Of 54 microscopically confirmed P. falciparum-infected subjects, PCR detected 90.7% (49/54). Out of 100 RDT-confirmed P. falciparum infections; PCR detected 80.0% (80/100). The sensitivity of PCR relative to microscopy and RDT was, therefore, 90.7% and 80%, respectively. The sensitivity of microscopy and RDT relative to PCR was 16.5 (49/299) and 24.2% (80/330), respectively. The overall PCR-based prevalence of P. falciparum infection was 5.6- and 3.3 fold higher than that determined by microscopy and RDT, respectively. None of the sub-microscopic subjects had severe anaemia, though 29.4% had mild anaemia (10–11.9 g/dl). Conclusions Asymptomatic, low-density malaria infection was common in the study area and PCR may be a better tool for measuring Plasmodium prevalence than microscopy and RDT. The inadequate sensitivity of the diagnostic methods to detect substantial number of sub-microscopic parasitaemia would undoubtedly affect malaria control efforts, making reduction of transmission more difficult. RDT and microscopy-based prevalence studies and subsequent reports of reduction in malaria incidence underestimate the true pictures of P. falciparum infections in the community. PCR, on the other hand, seems to have reasonable sensitivity to detect a higher number of infected subjects with low and sub-microscopic parasite densities than RDTs or microscopy. PMID:24090230

Detection of U.S., Lelystad, and European-like porcine reproductive and respiratory syndrome viruses and relative quantitation in boar semen and serum samples by real-time PCR.

PubMed

Wasilk, A; Callahan, J D; Christopher-Hennings, J; Gay, T A; Fang, Y; Dammen, M; Reos, M E; Torremorell, M; Polson, D; Mellencamp, M; Nelson, E; Nelson, W M

2004-10-01

Transmission of porcine reproductive and respiratory syndrome virus (PRRSV) via boar semen has been documented. Since semen is widely disseminated for artificial insemination and the virus can cause significant health and economic consequences, it is essential to have well-validated, rapid diagnostic techniques to detect and quantitate the virus for diagnostic and research purposes. Previously, boar semen was tested by a nested PCR (nPCR) assay which was compared to the "gold standard" swine bioassay. A correlation of 94% was observed, indicating that, most of the time, PCR detected infectious virus. Subsequently, a real-time PCR targeting the 3' untranslated region of the PRRSV genome was compared with nPCR by testing 413 serum and semen samples from PRRSV-inoculated and control boars. There was 95% agreement between the results of the two tests, with the majority of samples with discordant results containing virus at the lower range of detection by the assays. The virus in all samples was quantitated by using a standard curve obtained by serial dilution of an in vitro transcript. By using the in vitro transcript, the lower limit of sensitivity was observed to be approximately 33 copies/ml. Reactivity with a panel of more than 100 PRRSV isolates from various geographical regions in the United States was also documented. No reactivity with nine nonrelated swine viruses was noted. A real-time PCR was also developed for the detection of the European Lelystad virus and the European-like PRRSV now found in the United States. In six of six PRRSV-inoculated boars, peak levels of viremia occurred at 5 days postinoculation (DPI) and were most consistently detectable throughout 22 DPI. In five of six boars, PRRSV was shed in semen for 0 to 2 days during the first 10 DPI; however, one of six boars shed the virus in semen through 32 DPI. Therefore, in general, the concentration and duration of PRRSV shedding in semen did not correlate with the quantity or duration of virus in serum. These differences warrant further studies into the factors that prevent viral replication in the reproductive tract.

Development of a PCR Assay for Diagnosing Trematode (Opisthorchis and Haplorchis) Infections in Human Stools

PubMed Central

Kanda, Seiji; Laimanivong, Sakhone; Shimono, Takaki; Darcy, Andrew Waleluma; Phyaluanglath, Amphay; Mishima, Nobuyuki; Nishiyama, Toshimasa

2017-01-01

We developed a combined conventional polymerase chain reaction (PCR) and real-time PCR (qPCR)-based assay for detecting and discriminating between Opisthorchis viverrini and Haplorchis taichui parasite infections. The first PCR amplifies the mitochondrial cytochrome c oxidase subunit I (COI) genes of parasites, and differential diagnosis is achieved by performing qPCR with specific primers and SYBR Green I. The detection limit of the assay was found to be 2.0 × 102 plasmid copies in a test in which a stool sample was spiked with a single egg, which is equivalent to 5 eggs per gram (EPG). The testing of 34 clinical stool samples that had been demonstrated to contain “Opisthorchis-like” eggs by microscopy showed that the novel assay exhibited a sensitivity of 100% for “Opisthorchis-like” parasitic infections, and 71% and 91% of these samples were found to be infected with O. viverrini and H. taichui, respectively. A further four parasitic infections were diagnosed in the 16 negative samples, and the microscopic findings of these samples were confirmed to be false negatives by sequencing analysis. The assay also displayed high specificity during the testing of 10 samples containing other common parasites. The fact that our qPCR SYBR Green I–based assay detected submicroscopic traces of parasitic DNA and was able to differentiate between parasites that produce eggs with similar morphologies indicates that it has a good potential for development of diagnostic application to use in areas where multiple parasites coexist. PMID:27821695

Two Multiplex Real-Time PCR Assays to Detect and Differentiate Acinetobacter baumannii and Non- baumannii Acinetobacter spp. Carrying blaNDM, blaOXA-23-Like, blaOXA-40-Like, blaOXA-51-Like, and blaOXA-58-Like Genes

PubMed Central

Yang, Qiu; Rui, Yongyu

2016-01-01

Nosocomial infections caused by Acinetobacter spp. resistant to carbapenems are increasingly reported worldwide. Carbapenem-resistant Acinetobacter (CRA) is becoming a serious concern with increasing patient morbidity, mortality, and lengths of hospital stay. Therefore, the rapid detection of CRA is essential for epidemiological surveillance. Polymerase chain reaction (PCR) has been extensively used for the rapid identification of most pathogens. In this study, we have developed two multiplex real-time PCR assays to detect and differentiate A. baumannii and non-A. baumannii Acinetobacter spp, and common carbapenemase genes, including blaNDM, blaOXA-23-like, blaOXA-40-like, blaOXA-51-like, and blaOXA-58-like. We demonstrate the potential utility of these assays for the direct detection of blaNDM-, blaOXA-23-like-, blaOXA-40-like-, blaOXA-51-like-, and blaOXA-58-like-positive CRA in clinical specimens. Primers were specifically designed, and two multiplex real-time PCR assays were developed: multiplex real-time PCR assay1 for the detection of Acinetobacter baumannii 16S–23S rRNA internal transcribed spacer sequence, the Acinetobacter recA gene, and class-B-metalloenzyme-encoding gene blaNDM; and multiplex real-time PCR assay2 to detect class-D-oxacillinase-encoding genes (blaOXA-23-like, blaOXA-40-like, blaOXA-51-like,and blaOXA-58-like). The assays were performed on an ABI Prism 7500 FAST Real-Time PCR System. CRA isolates were used to compare the assays with conventional PCR and sequencing. Known amounts of CRA cells were added to sputum and fecal specimens and used to test the multiplex real-time PCR assays. The results for target and nontarget amplification showed that the multiplex real-time PCR assays were specific, the limit of detection for each target was 10 copies per 20 μL reaction volume, the assays were linear over six log dilutions of the target genes (r2 > 0.99), and the Ct values of the coefficients of variation for intra- and interassay reproducibility were less than 5%. The multiplex real-time PCR assays showed 100% concordance with conventional PCR when tested against 400 CRA isolates and their sensitivity for the target DNA in sputum and fecal specimens was 102 CFU/mL. Therefore, these novel multiplex real-time PCR assays allow the sensitive and specific characterization and differentiation of blaNDM-, blaOXA-23-like-, blaOXA-40-like-, blaOXA-51-like-, and blaOXA-58-like-positive CRA, making them potential tools for the direct detection of CRA in clinical specimens and the surveillance of nosocomial infections. PMID:27391234

Comparison of PCR-Electrospray Ionization Mass Spectrometry with 16S rRNA PCR and Amplicon Sequencing for Detection of Bacteria in Excised Heart Valves

PubMed Central

Peeters, Bart; Herijgers, Paul; Beuselinck, Kurt; Peetermans, Willy E.; Herregods, Marie-Christin

2016-01-01

Identification of the causative pathogen of infective endocarditis (IE) is crucial for adequate management and therapy. A broad-range PCR-electrospray ionization mass spectrometry (PCR-ESI-MS) technique was compared with broad-spectrum 16S rRNA PCR and amplicon sequencing (16S rRNA PCR) for the detection of bacterial pathogens in 40 heart valves obtained from 34 definite infective endocarditis patients according to the modified Duke criteria and six nonendocarditis patients. Concordance between the two molecular techniques was 98% for being positive or negative, 97% for concordant identification up to the genus level, and 77% for concordant identification up to the species level. Sensitivity for detecting the causative pathogen (up to the genus level) in excised heart valves was 88% for 16S rRNA PCR and 85% for PCR-ESI-MS; the specificity was 83% for both methods. The two molecular techniques were significantly more sensitive than valve culture (18%) and accurately identified bacteria in excised heart valves. In eight patients with culture-negative IE, the following results were obtained: concordant detection of Coxiella burnetii (n = 2), Streptococcus gallolyticus (n = 1), Propionibacterium acnes (n = 1), and viridans group streptococci (n = 1) by both molecular tests, detection of P. acnes by PCR-ESI-MS whereas the 16S rRNA PCR was negative (n = 1), and a false-negative result by both molecular techniques (n = 2). In one case of IE caused by viridans streptococci, PCR-ESI-MS was positive for Enterococcus spp. The advantages of PCR-ESI-MS compared to 16S rRNA PCR are its automated workflow and shorter turnaround times. PMID:27629895

Evaluation of the Loop Mediated Isothermal DNA Amplification (LAMP) Kit for Malaria Diagnosis in P. vivax Endemic Settings of Colombia

PubMed Central

Vallejo, Andrés F.; Martínez, Nora L.; González, Iveth J.; Arévalo-Herrera, Myriam; Herrera, Sócrates

2015-01-01

Background Most commonly used malaria diagnostic tests, including microscopy and antigen-detecting rapid tests, cannot reliably detect low-density infections which are frequent in low transmission settings. Molecular methods such as polymerase chain reaction (PCR) are highly sensitive but remain too laborious for field deployment. In this study, the applicability of a malaria diagnosis kit based on loop-mediated isothermal amplification (mLAMP) was assessed in malaria endemic areas of Colombia with Plasmodium vivax predominance. Methodology/Principal Findings First, a passive case detection (PCD) study on 278 febrile patients recruited in Tierralta (department of Cordoba) was conducted to assess the diagnostic performance of the mLAMP method. Second, an active case detection (ACD) study on 980 volunteers was conducted in 10 sentinel sites with different epidemiological profiles. Whole blood samples were processed for microscopic and mLAMP diagnosis. Additionally RT-PCR and nested RT-PCR were used as reference tests. In the PCD study, P. falciparum accounted for 23.9% and P. vivax for 76.1% of the infections and no cases of mixed-infections were identified. Microscopy sensitivity for P. falciparum and P. vivax were 100% and 86.1%, respectively. mLAMP sensitivity for P. falciparum and P. vivax was 100% and 91.4%, respectively. In the ACD study, mLAMP detected 65 times more cases than microscopy. A high proportion (98.0%) of the infections detected by mLAMP was from volunteers without symptoms. Conclusions/Significance mLAMP sensitivity and specificity were comparable to RT-PCR. LAMP was significantly superior to microscopy and in P. vivax low-endemicity settings and under minimum infrastructure conditions, it displayed sensitivity and specificity similar to that of single-well RT-PCR for detection of both P. falciparum and P. vivax infections. Here, the dramatically increased detection of asymptomatic malaria infections by mLAMP demonstrates the usefulness of this new tool for diagnosis, surveillance, and screening in elimination strategies. PMID:25569550

Quantitative polymerase chain reaction (PCR) for detection of aquatic animal pathogens in a diagnostic laboratory setting

USGS Publications Warehouse

Purcell, Maureen K.; Getchell, Rodman G.; McClure, Carol A.; Weber, S.E.; Garver, Kyle A.

2011-01-01

Real-time, or quantitative, polymerase chain reaction (qPCR) is quickly supplanting other molecular methods for detecting the nucleic acids of human and other animal pathogens owing to the speed and robustness of the technology. As the aquatic animal health community moves toward implementing national diagnostic testing schemes, it will need to evaluate how qPCR technology should be employed. This review outlines the basic principles of qPCR technology, considerations for assay development, standards and controls, assay performance, diagnostic validation, implementation in the diagnostic laboratory, and quality assurance and control measures. These factors are fundamental for ensuring the validity of qPCR assay results obtained in the diagnostic laboratory setting.

Construction and Evaluation of Internal Control DNA for PCR Amplification of Chlamydia trachomatis DNA from Urine Samples

PubMed Central

Betsou, Fotini; Beaumont, Katy; Sueur, Jean Marie; Orfila, Jeanne

2003-01-01

An internal control DNA (ICD) with the same primer binding sequences as the target Chlamydia trachomatis DNA was constructed and evaluated in a PCR assay with immunoenzymatic detection. One hundred urine specimens were tested, and 23 were found to contain inhibitors of the PCR, if not subjected to DNA extraction prior to amplification. Coamplification and detection of the ICD appeared to be a useful method for estimating the effects of inhibitors on C. trachomatis DNA amplification. PMID:12624066

Molecular diagnosis of canine visceral leishmaniasis: a comparative study of three methods using skin and spleen from dogs with natural Leishmania infantum infection.

PubMed

Reis, Levi Eduardo Soares; Coura-Vital, Wendel; Roatt, Bruno Mendes; Bouillet, Leoneide Érica Maduro; Ker, Henrique Gama; Fortes de Brito, Rory Cristiane; Resende, Daniela de Melo; Carneiro, Mariângela; Giunchetti, Rodolfo Cordeiro; Marques, Marcos José; Carneiro, Cláudia Martins; Reis, Alexandre Barbosa

2013-11-08

Polymerase chain reaction (PCR) and its variations represent highly sensitive and specific methods for Leishmania DNA detection and subsequent canine visceral leishmaniasis (CVL) diagnosis. The aim of this work was to compare three different molecular diagnosis techniques (conventional PCR [cPCR], seminested PCR [snPCR], and quantitative PCR [qPCR]) in samples of skin and spleen from 60 seropositive dogs by immunofluorescence antibody test and enzyme-linked immunosorbent assay. Parasitological analysis was conducted by culture of bone marrow aspirate and optical microscopic assessment of ear skin and spleen samples stained with Giemsa, the standard tests for CVL diagnosis. The primers L150/L152 and LINR4/LIN17/LIN19 were used to amplify the conserved region of the Leishmania kDNA minicircle in the cPCR, and snPCR and qPCR were performed using the DNA polymerase gene (DNA pol α) primers from Leishmania infantum. The parasitological analysis revealed parasites in 61.7% of the samples. Sensitivities were 89.2%, 86.5%, and 97.3% in the skin and 81.1%, 94.6%, and 100.0% in spleen samples used for cPCR, snPCR, and qPCR, respectively. We demonstrated that the qPCR method was the best technique to detect L. infantum in both skin and spleen samples. However, we recommend the use of skin due to the high sensitivity and sampling being less invasive. Copyright © 2013 Elsevier B.V. All rights reserved.

Development of multiplex PCR for the detection of total coliform bacteria for Escherichia coli and Clostridium perfringens in drinking water.

PubMed

Tantawiwat, Suwalee; Tansuphasiri, Unchalee; Wongwit, Waranya; Wongchotigul, Varee; Kitayaporn, Dwip

2005-01-01

Multiplex PCR amplification of lacZ, uidA and plc genes was developed for the simultaneous detection of total coliform bacteria for Escherichia coli and Clostridium perfringens, in drinking water. Detection by agarose gel electrophoresis yielded a band of 876 bp for the lacZ gene of all coliform bacteria; a band of 147 bp for the uidA gene and a band of 876 bp for the lacZ gene of all strains of E. coli; a band of 280 bp for the p/c gene for all strains of C. perfringens; and a negative result for all three genes when tested with other bacteria. The detection limit was 100 pg for E. coli and C. perfringens, and 1 ng for coliform bacteria when measured with purified DNA. This assay was applied to the detection of these bacteria in spiked water samples. Spiked water samples with 0-1,000 CFU/ml of coliform bacteria and/or E. coli and/or C. perfringens were detected by this multiplex PCR after a pre-enrichment step to increase the sensitivity and to ensure that the detection was based on the presence of cultivable bacteria. The result of bacterial detection from the multiplex PCR was comparable with that of a standard plate count on selective medium (p=0.62). When using standard plate counts as a gold standard, the sensitivity for this test was 99.1% (95% CI 95.33, 99.98) and the specificity was 90.9 % (95% CI 75.67, 98.08). Multiplex PCR amplification with a pre-enrichment step was shown to be an effective, sensitive and rapid method for the simultaneous detection of these three microbiological parameters in drinking water.

Exhaust Air Dust Monitoring is Superior to Soiled Bedding Sentinels for the Detection of Pasteurella pneumotropica in Individually Ventilated Cage Systems.

PubMed

Miller, Manuel; Ritter, Brbel; Zorn, Julia; Brielmeier, Markus

2016-11-01

Reliable detection of unwanted organisms is essential for meaningful health monitoring in experimental animal facilities. Currently, most rodents are housed in IVC systems, which prevent the aerogenic transmission of pathogens between cages. Typically soiled-bedding sentinels (SBS) exposed to soiled bedding collected from a population of animals within an IVC rack are tested as representatives, but infectious agents often go undetected due to inefficient transmission. Pasteurellaceae are among the most prevalent bacterial pathogens isolated from experimental mice, and the failure of SBS to detect these bacteria is well established. In this study, we investigated whether analysis of exhaust air dust (EAD) samples by using a sensitive and specific real-time PCR assay is superior to conventional SBS monitoring for the detection of Pasteurella pneumotropica (Pp) infections. In a rack with a known prevalence of Pp-positive mice, weekly EAD sampling was compared with the classic SBS method over 3 mo. In 6 rounds of testing, with a prevalence of 5 infected mice in each of 7 cages in a rack of 63 cages, EAD PCR detected Pp at every weekly time point; SBS failed to detect Pp in all cases. The minimal prevalence of Pp-infected mice required to obtain a reliable positive result by EAD PCR testing was determined to be 1 in 63 cages. Reliable detection of Pp was achieved after only 1 wk of exposure. Analysis of EAD samples by real-time PCR assay provides a sensitive, simple, and reliable approach for Pp identification in laboratory mice.

Exhaust Air Dust Monitoring is Superior to Soiled Bedding Sentinels for the Detection of Pasteurella pneumotropica in Individually Ventilated Cage Systems

PubMed Central

Miller, Manuel; Ritter, Bärbel; Zorn, Julia; Brielmeier, Markus

2016-01-01

Reliable detection of unwanted organisms is essential for meaningful health monitoring in experimental animal facilities. Currently, most rodents are housed in IVC systems, which prevent the aerogenic transmission of pathogens between cages. Typically soiled-bedding sentinels (SBS) exposed to soiled bedding collected from a population of animals within an IVC rack are tested as representatives, but infectious agents often go undetected due to inefficient transmission. Pasteurellaceae are among the most prevalent bacterial pathogens isolated from experimental mice, and the failure of SBS to detect these bacteria is well established. In this study, we investigated whether analysis of exhaust air dust (EAD) samples by using a sensitive and specific real-time PCR assay is superior to conventional SBS monitoring for the detection of Pasteurella pneumotropica (Pp) infections. In a rack with a known prevalence of Pp-positive mice, weekly EAD sampling was compared with the classic SBS method over 3 mo. In 6 rounds of testing, with a prevalence of 5 infected mice in each of 7 cages in a rack of 63 cages, EAD PCR detected Pp at every weekly time point; SBS failed to detect Pp in all cases. The minimal prevalence of Pp-infected mice required to obtain a reliable positive result by EAD PCR testing was determined to be 1 in 63 cages. Reliable detection of Pp was achieved after only 1 wk of exposure. Analysis of EAD samples by real-time PCR assay provides a sensitive, simple, and reliable approach for Pp identification in laboratory mice. PMID:27931316

Broad-range PCR coupled with mass-spectrometry for the detection of Mycobacterium tuberculosis drug resistance

PubMed Central

Florea, Dragoş; Oţelea, Dan; Olaru, Ioana D.; Hristea, Adriana

2016-01-01

Background The need to limit the spread of drug-resistant Mycobacterium tuberculosis requires rapid detection of resistant strains. The present study aimed to evaluate a commercial assay using broad-range PCR coupled with electrospray ionization mass spectrometry (PCR/ESI-MS) for the rapid detection of isoniazid (INH) and rifampin (RIF) resistance in M. tuberculosis strains isolated from Romanian patients with pulmonary tuberculosis. Methods PCR/ESI-MS was used to detect genotypic resistance to RIF and INH in a panel of 63 M. tuberculosis isolates phenotypically characterized using the absolute concentration method on Löwenstein-Jensen medium. Results Thirty-eight (60%) strains were susceptible to both drugs, 22 (35%) were RIF and INH resistant, one was INH mono-resistant and two were RIF mono-resistant. The sensitivity for INH and RIF resistance mutations detection were 100% and 92% respectively, with a specificity of more than 95% for each drug. Conclusion PCR/ESI-MS is a good method for the detection of RIF and INH resistance and might represent an alternative to other rapid diagnostic tests for the detection of genetic markers of resistance in M. tuberculosis isolates. PMID:27019827

A new detection method for the K variant of butyrylcholinesterase based on PCR primer introduced restriction analysis (PCR-PIRA).

PubMed Central

Shibuta, K; Abe, M; Suzuki, T

1994-01-01

The K variant of human butyrylcholinesterase is caused by a G/A transition in the butyrylcholinesterase gene, which neither creates nor destroys any restriction site. In an attempt to detect the K variant both simply and rapidly, we developed a two step method of "PCR primer introduced restriction analysis" (PCR-PIRA). The first step was used to introduce a new Fun4HI site into the normal allele for a screening test, while the second step was performed to create a new MaeIII site on the variant allele for a specific test. This method thus enabled us to distinguish clearly the K variant from the normal allele, and also showed that the frequency of the K variant allele is 0.164 in the Japanese population. Images PMID:7966197

Performance of four HRP-2/pLDH combination rapid diagnostic tests and field microscopy as screening tests for malaria in pregnancy in Indonesia: a cross-sectional study.

PubMed

Ahmed, Rukhsana; Levy, Elvira I; Maratina, Sylvia S; de Jong, Judith J; Asih, Puji B S; Rozi, Ismail E; Hawley, William; Syafruddin, Din; ter Kuile, Feiko

2015-10-29

Malaria in pregnancy poses a major public health problem in Indonesia with an estimated six million pregnancies at risk of Plasmodium falciparum or Plasmodium vivax malaria annually. In 2010, Indonesia introduced a screen and treat policy for the control of malaria in pregnancy at first antenatal visit using microscopy or rapid diagnostic tests (RDTs). A diagnostic study was conducted in Sumba, Indonesia to compare the performance of four different RDTs in predominately asymptomatic pregnant women under field condition. Women were screened for malaria at antenatal visits using field microscopy and four HRP-2/pLDH combination RDTs (Carestart™, First-Response(®), Parascreen(®) and SD-Bioline(®)). The test results were compared with expert microscopy and nested PCR. End user experience of the RDTs in the field was assessed by questionnaire. Overall 950 were recruited and 98.7 % were asymptomatic. The prevalence of malaria was 3.0-3.4 % by RDTs, and 3.6, 5.0 and 6.6 % by field microscopy, expert microscopy and PCR, respectively. The geometric-mean parasite density was low (P. falciparum = 418, P. vivax = 147 parasites/µL). Compared with PCR, the overall sensitivity of the RDTs and field microscopy to detect any species was 24.6-31.1 %; specificities were >98.4 %. Relative to PCR, First-Response(®) had the best diagnostic accuracy (any species): sensitivity = 31.1 %, specificity = 98.9 % and diagnostic odds ratio = 39.0 (DOR). The DOR values for Carestart™, Parascreen(®), SD-Bioline(®), and field microscopy were 23.4, 23.7, 23.5 and 29.2, respectively. The sensitivity of Pan-pLDH bands to detect PCR confirmed P. vivax mono-infection were 8.6-13.0 %. The sensitivity of the HRP-2 band alone to detect PCR confirmed P. falciparum was 10.3-17.9 %. Pan-pLDH detected P. falciparum cases undetected by the HRP-2 band resulting in a better test performance when both bands were combined. First Response(®) was preferred by end-users for the overall practicality. The diagnostic accuracy to detect malaria among mostly asymptomatic pregnant women and perceived ease of use was slightly better with First-Response(®), but overall, differences between the four RDTs were small and performance comparable to field microscopy. Combination RDTs are a suitable alternative to field microscopy to screen for malaria in pregnancy in rural Indonesia. The clinical relevance of low density malaria infections detected by PCR, but undetected by RDTs or microscopy needs to be determined.

Continuous-flow, microfluidic, qRT-PCR system for RNA virus detection.

PubMed

Fernández-Carballo, B Leticia; McBeth, Christine; McGuiness, Ian; Kalashnikov, Maxim; Baum, Christoph; Borrós, Salvador; Sharon, Andre; Sauer-Budge, Alexis F

2018-01-01

One of the main challenges in the diagnosis of infectious diseases is the need for rapid and accurate detection of the causative pathogen in any setting. Rapid diagnosis is key to avoiding the spread of the disease, to allow proper clinical decisions to be made in terms of patient treatment, and to mitigate the rise of drug-resistant pathogens. In the last decade, significant interest has been devoted to the development of point-of-care reverse transcription polymerase chain reaction (PCR) platforms for the detection of RNA-based viral pathogens. We present the development of a microfluidic, real-time, fluorescence-based, continuous-flow reverse transcription PCR system. The system incorporates a disposable microfluidic chip designed to be produced industrially with cost-effective roll-to-roll embossing methods. The chip has a long microfluidic channel that directs the PCR solution through areas heated to different temperatures. The solution first travels through a reverse transcription zone where RNA is converted to complementary DNA, which is later amplified and detected in real time as it travels through the thermal cycling area. As a proof of concept, the system was tested for Ebola virus detection. Two different master mixes were tested, and the limit of detection of the system was determined, as was the maximum speed at which amplification occurred. Our results and the versatility of our system suggest its promise for the detection of other RNA-based viruses such as Zika virus or chikungunya virus, which constitute global health threats worldwide. Graphical abstract Photograph of the RT-PCR thermoplastic chip.

Detection of Alicyclobacillus spp. in Fruit Juice by Combination of Immunomagnetic Separation and a SYBR Green I Real-Time PCR Assay

PubMed Central

Yuan, Yahong; Liu, Bin; Wang, Ling; Yue, Tianli

2015-01-01

An approach based on immunomagnetic separation (IMS) and SYBR Green I real-time PCR (real-time PCR) with species-specific primers and melting curve analysis was proposed as a rapid and effective method for detecting Alicyclobacillus spp. in fruit juices. Specific primers targeting the 16S rDNA sequences of Alicyclobacillus spp. were designed and then confirmed by the amplification of DNA extracted from standard strains and isolates. Spiked samples containing known amounts of target bacteria were used to obtain standard curves; the correlation coefficient was greater than 0.986 and the real-time PCR amplification efficiencies were 98.9%- 101.8%. The detection limit of the testing system was 2.8×101 CFU/mL. The coefficient of variation for intra-assay and inter-assay variability were all within the acceptable limit of 5%. Besides, the performance of the IMS-real-time PCR assay was further investigated by detecting naturally contaminated kiwi fruit juice; the sensitivity, specificity and accuracy were 91.7%, 95.9% and 95.3%, respectively. The established IMS-real-time PCR procedure provides a new method for identification and quantitative detection of Alicyclobacillus spp. in fruit juice. PMID:26488469

Multilaboratory comparison of hepatitis C virus viral load assays.

PubMed

Caliendo, A M; Valsamakis, A; Zhou, Y; Yen-Lieberman, B; Andersen, J; Young, S; Ferreira-Gonzalez, A; Tsongalis, G J; Pyles, R; Bremer, J W; Lurain, N S

2006-05-01

We report a multilaboratory evaluation of hepatitis C virus (HCV) viral load assays to determine their linear range, reproducibility, subtype detection, and agreement. A panel of HCV RNA samples ranging in nominal concentration from 1.0 to 7.0 log10 IU/ml was constructed by diluting a clinical specimen (genotype 1b). Replicates of the panel were tested in multiple laboratories using the Abbott TaqMan analyte-specific reagent (Abbott reverse transcription-PCR [RT-PCR]), Roche TaqMan RUO (Roche RT-PCR), Roche Amplicor Monitor HCV 2.0 (Roche Monitor), and Bayer VERSANT HCV RNA 3.0 (Bayer bDNA) assays. Bayer bDNA-negative specimens were tested reflexively using the Bayer VERSANT HCV RNA qualitative assay (Bayer TMA). Abbott RT-PCR and Roche RT-PCR detected all 28 replicates with a concentration of 1.0 log10 IU/ml and were linear to 7.0 log10 IU/ml. Roche Monitor and Bayer bDNA detected 27 out of 28 and 13 out of 28 replicates, respectively, of 3.0 log10 IU/ml. Bayer TMA detected all seven replicates with 1.0 log10 IU/ml. Bayer bDNA was the most reproducible of the four assays. The mean viral load values for panel members in the linear ranges of the assays were within 0.5 log10 for the different tests. Eighty-nine clinical specimens of various genotypes (1 through 4) were tested in the Bayer bDNA, Abbott RT-PCR, and Roche RT-PCR assays. For Abbott RT-PCR, mean viral load values were 0.61 to 0.96 log10 greater than the values for Bayer bDNA assay for samples with genotype 1, 2, or 3 samples and 0.08 log10 greater for genotype 4 specimens. The Roche RT-PCR assay gave mean viral load values that were 0.28 to 0.82 log10 greater than those obtained with the Bayer bDNA assay for genotype 1, 2, and 3 samples. However, for genotype 4 samples the mean viral load value obtained with the Roche RT-PCR assay was, on average, 0.15 log10 lower than that of the Bayer bDNA. Based on these data, we conclude that the sensitivity and linear range of the Abbott and Roche RT-PCR assays enable them to be used for HCV diagnostics and therapeutic monitoring. However, the differences in the viral load values obtained with the different assays underscore the importance of using one assay when monitoring response to therapy.

Sensitivity of Small RNA-Based Detection of Plant Viruses.

PubMed

Santala, Johanna; Valkonen, Jari P T

2018-01-01

Plants recognize unrelated viruses by the antiviral defense system called RNA interference (RNAi). RNAi processes double-stranded viral RNA into small RNAs (sRNAs) of 21-24 nucleotides, the reassembly of which into longer strands in silico allows virus identification by comparison with the sequences available in databases. The aim of this study was to compare the virus detection sensitivity of sRNA-based virus diagnosis with the established virus species-specific polymerase chain reaction (PCR) approach. Viruses propagated in tobacco plants included three engineered, infectious clones of Potato virus A (PVA), each carrying a different marker gene, and an infectious clone of Potato virus Y (PVY). Total RNA (containing sRNA) was isolated and subjected to reverse-transcription real-time PCR (RT-RT-PCR) and sRNA deep-sequencing at different concentrations. RNA extracted from various crop plants was included in the reactions to normalize RNA concentrations. Targeted detection of selected viruses showed a similar threshold for the sRNA and reverse-transcription quantitative PCR (RT-qPCR) analyses. The detection limit for PVY and PVA by RT-qPCR in this study was 3 and 1.5 fg of viral RNA, respectively, in 50 ng of total RNA per PCR reaction. When knowledge was available about the viruses likely present in the samples, sRNA-based virus detection was 10 times more sensitive than RT-RT-PCR. The advantage of sRNA analysis is the detection of all tested viruses without the need for virus-specific primers or probes.

Microtiter format for simultaneous multianalyte detection and development of a PCR-chemiluminescent enzyme immunoassay for typing human papillomavirus DNAs.

PubMed

Roda, Aldo; Mirasoli, Mara; Venturoli, Simona; Cricca, Monica; Bonvicini, Francesca; Baraldini, Mario; Pasini, Patrizia; Zerbini, Marialuisa; Musiani, Monica

2002-10-01

To allow multianalyte binding assays, we have developed a novel polystyrene microtiter plate containing 24 main wells, each divided into 7 subwells. We explored its clinical potential by developing a PCR-chemiluminescent immunoassay (PCR-CLEIA) for simultaneous detection and typing of seven high oncogenic risk human papillomavirus (HPV) DNAs in one well. Seven different oligonucleotide probes, each specific for a high-risk HPV genotype, were separately immobilized in the subwells. Subsequently, a digoxigenin-labeled consensus PCR amplification product was added to the main well. The PCR product hybridized to the immobilized probe corresponding to its genotype and was subsequently detected by use of a peroxidase-labeled anti-digoxigenin antibody and chemiluminescence imaging with an ultrasensitive charge-coupled device camera. Results obtained for 50 cytologic samples were compared with those obtained with a conventional colorimetric PCR-ELISA. The method was specific and allowed detection of 50 genome copies of HPV 16, 18, 33, and 58, and 100 genome copies of HPV 31, 35, and 45. Intra- and interassay CVs for the method were 5.6% and 7.9%, respectively. All results obtained for clinical samples were confirmed by the conventional PCR-ELISA. PCR-CLEIA allows rapid, single-tube simultaneous detection and typing of seven high-risk HPV DNAs with small reagent volumes. The principle appears applicable to the development of other single-tube panels of tests.

Rapid detection of Listeria monocytogenes in foods, by a combination of PCR and DNA probe.

PubMed

Ingianni, A; Floris, M; Palomba, P; Madeddu, M A; Quartuccio, M; Pompei, R

2001-10-01

Listeria monocytogenes is a frequent contaminant of water and foods. Its rapid detection is needed before some foods can be prepared for marketing. In this work L. monocytogenes has been searched for in foods, by a combination of polymerase chain reaction (PCR) and a DNA probe. Both PCR and the probe were prepared for recognizing a specific region of the internalin gene, which is responsible for the production of one of the most important pathogenic factors of Listeria. The combined use of PCR and the DNA probe was used for the detection of L. monocytogenes in over 180 environmental and food samples. Several detection methods were compared in this study, namely conventional culture methods; direct PCR; PCR after an enrichment step; a DNA probe alone; a DNA probe after enrichment and another commercially available gene-probe. Finally PCR and the DNA probe were used in series on all the samples collected. When the DNA probe was associated with the PCR, specific and accurate detection of listeria in the samples could be obtained in about a working-day. The present molecular method showed some advantages in terms of rapidity and specificity in comparison to the other aforementioned tests. In addition, it resulted as being easy to handle, even for non-specialized personnel in small diagnostic microbiology laboratories. Copyright 2001 Academic Press.

Development and inter-laboratory validation study of an improved new real-time PCR assay with internal control for detection and laboratory diagnosis of African swine fever virus.

PubMed

Tignon, Marylène; Gallardo, Carmina; Iscaro, Carmen; Hutet, Evelyne; Van der Stede, Yves; Kolbasov, Denis; De Mia, Gian Mario; Le Potier, Marie-Frédérique; Bishop, Richard P; Arias, Marisa; Koenen, Frank

2011-12-01

A real-time polymerase chain reaction (PCR) assay for the rapid detection of African swine fever virus (ASFV), multiplexed for simultaneous detection of swine beta-actin as an endogenous control, has been developed and validated by four National Reference Laboratories of the European Union for African swine fever (ASF) including the European Union Reference Laboratory. Primers and a TaqMan(®) probe specific for ASFV were selected from conserved regions of the p72 gene. The limit of detection of the new real-time PCR assay is 5.7-57 copies of the ASFV genome. High accuracy, reproducibility and robustness of the PCR assay (CV ranging from 0.7 to 5.4%) were demonstrated both within and between laboratories using different real-time PCR equipments. The specificity of virus detection was validated using a panel of 44 isolates collected over many years in various geographical locations in Europe, Africa and America, including recent isolates from the Caucasus region, Sardinia, East and West Africa. Compared to the OIE-prescribed conventional and real-time PCR assays, the sensitivity of the new assay with internal control was improved, as demonstrated by testing 281 field samples collected in recent outbreaks and surveillance areas in Europe and Africa (170 samples) together with samples obtained through experimental infections (111 samples). This is particularly evident in the early days following experimental infection and during the course of the disease in pigs sub-clinically infected with strains of low virulence (from 35 up to 70dpi). The specificity of the assay was also confirmed on 150 samples from uninfected pigs and wild boar from ASF-free areas. Measured on the total of 431 tested samples, the positive deviation of the new assay reaches 21% or 26% compared to PCR and real-time PCR methods recommended by OIE. This improved and rigorously validated real-time PCR assay with internal control will provide a rapid, sensitive and reliable molecular tool for ASFV detection in pigs in newly infected areas, control in endemic areas and surveillance in ASF-free areas. Copyright © 2011 Elsevier B.V. All rights reserved.

Development and use of tuf gene-based primers for the multiplex PCR detection of Lactobacillus acidophilus, Lactobacillus casei group, Lactobacillus delbrueckii, and Bifidobacterium longum in commercial dairy products.

PubMed

Sheu, Sen-Je; Hwang, Wen-zhe; Chen, Hsin-Chih; Chiang, Yu-Cheng; Tsen, Hau-Yang

2009-01-01

PCR primers specific for the detection of Lactobacillus acidophilus, Lactobacillus casei group, Lactobacillus delbrueckii, and Bifidobacterium longum were designed based on the elongation factor Tu gene (tuf). The specificity of these four primer sets were confirmed by PCR with 88 bacterial strains of Lactobacillus, Enterococcus, Bifidobacterium, and other bacterial species. Results indicated that these primer sets generated predicted PCR products of 397, 230, 202, and 161 bp for L. acidophilus, L. delbrueckii, L. casei group, and B. longum, respectively. Bacterial species other than the target organisms tested did not generate false-positive results. When these four primer sets were combined for the simultaneous detection of the lactic acid bacteria (LAB) in fermented milk products including yogurt, the LAB species listed on the labels of these products could be identified without the preenrichment step. The identification limit for each LAB strain with this multiplex PCR method was N X 10(3) CFU/ml in milk samples. The results of our multiplex PCR method were confirmed by PCR assay using primers based on the 16S rDNA or the 16S-23S intergenic spacer region and by biochemical tests using the API 50 CHL kit. When this multiplex PCR method was used with the determination of counts of total viable LAB and bifidobacteria, the quality of commercial fermented milk products could be assured.

Comparison of ELISA, nested PCR and sequencing and a novel qPCR for detection of Giardia isolates from Jordan.

PubMed

Hijjawi, Nawal; Yang, Rongchang; Hatmal, Ma'mon; Yassin, Yasmeen; Mharib, Taghrid; Mukbel, Rami; Mahmoud, Sameer Alhaj; Al-Shudifat, Abdel-Ellah; Ryan, Una

2018-02-01

Little is known about the prevalence of Giardia duodenalis in human patients in Jordan and all previous studies have used direct microscopy, which lacks sensitivity. The present study developed a novel quantitative PCR (qPCR) assay at the β-giardin (bg) locus and evaluated its use as a frontline test for the diagnosis of giardiasis in comparison with a commercially available ELISA using nested PCR and sequencing of the glutamate dehydrogenase (gdh) locus (gdh nPCR) as the gold standard. A total of 96 human faecal samples were collected from 96 patients suffering from diarrhoea from 5 regions of Jordan and were screened using the ELISA and qPCR. The analytical specificity of the bg qPCR assay revealed no cross-reactions with other genera and detected all the Giardia isolates tested. Analytical sensitivity was 1 Giardia cyst per μl of DNA extract. The overall prevalence of Giardia was 64.6%. The clinical sensitivity and specificity of the bg qPCR was 89.9% and 82.9% respectively compared to 76.5 and 68.0% for the ELISA. This study is the first to compare three different methods (ELISA, bg qPCR, nested PCR and sequencing at the gdh locus) to diagnose Jordanian patients suffering from giardiasis and to analyze their demographic data. Copyright © 2018 Elsevier Inc. All rights reserved.

Cross-platform evaluation of commercial real-time SYBR green RT-PCR kits for sensitive and rapid detection of European bat Lyssavirus type 1.

PubMed

Picard-Meyer, Evelyne; Peytavin de Garam, Carine; Schereffer, Jean Luc; Marchal, Clotilde; Robardet, Emmanuelle; Cliquet, Florence

2015-01-01

This study evaluates the performance of five two-step SYBR Green RT-qPCR kits and five one-step SYBR Green qRT-PCR kits using real-time PCR assays. Two real-time thermocyclers showing different throughput capacities were used. The analysed performance evaluation criteria included the generation of standard curve, reaction efficiency, analytical sensitivity, intra- and interassay repeatability as well as the costs and the practicability of kits, and thermocycling times. We found that the optimised one-step PCR assays had a higher detection sensitivity than the optimised two-step assays regardless of the machine used, while no difference was detected in reaction efficiency, R (2) values, and intra- and interreproducibility between the two methods. The limit of detection at the 95% confidence level varied between 15 to 981 copies/µL and 41 to 171 for one-step kits and two-step kits, respectively. Of the ten kits tested, the most efficient kit was the Quantitect SYBR Green qRT-PCR with a limit of detection at 95% of confidence of 20 and 22 copies/µL on the thermocyclers Rotor gene Q MDx and MX3005P, respectively. The study demonstrated the pivotal influence of the thermocycler on PCR performance for the detection of rabies RNA, as well as that of the master mixes.

Cross-Platform Evaluation of Commercial Real-Time SYBR Green RT-PCR Kits for Sensitive and Rapid Detection of European Bat Lyssavirus Type 1

PubMed Central

Picard-Meyer, Evelyne; Peytavin de Garam, Carine; Schereffer, Jean Luc; Marchal, Clotilde; Robardet, Emmanuelle; Cliquet, Florence

2015-01-01

This study evaluates the performance of five two-step SYBR Green RT-qPCR kits and five one-step SYBR Green qRT-PCR kits using real-time PCR assays. Two real-time thermocyclers showing different throughput capacities were used. The analysed performance evaluation criteria included the generation of standard curve, reaction efficiency, analytical sensitivity, intra- and interassay repeatability as well as the costs and the practicability of kits, and thermocycling times. We found that the optimised one-step PCR assays had a higher detection sensitivity than the optimised two-step assays regardless of the machine used, while no difference was detected in reaction efficiency, R 2 values, and intra- and interreproducibility between the two methods. The limit of detection at the 95% confidence level varied between 15 to 981 copies/µL and 41 to 171 for one-step kits and two-step kits, respectively. Of the ten kits tested, the most efficient kit was the Quantitect SYBR Green qRT-PCR with a limit of detection at 95% of confidence of 20 and 22 copies/µL on the thermocyclers Rotor gene Q MDx and MX3005P, respectively. The study demonstrated the pivotal influence of the thermocycler on PCR performance for the detection of rabies RNA, as well as that of the master mixes. PMID:25785274

Comparing the Yield of Nasopharyngeal Swabs, Nasal Aspirates, and Induced Sputum for Detection of Bordetella pertussis in Hospitalized Infants.

PubMed

Nunes, Marta C; Soofie, Nasiha; Downs, Sarah; Tebeila, Naume; Mudau, Azwi; de Gouveia, Linda; Madhi, Shabir A

2016-12-01

 Advances in molecular laboratory techniques are changing the landscape of Bordetella pertussis illness diagnosis. Polymerase chain reaction (PCR) assays have greatly improved the sensitivity detection and the turnaround time to diagnosis compared to culture. Moreover, different respiratory specimens, such as flocked nasopharyngeal swabs (NPSs), nasopharyngeal aspirates (NPAs), and induced sputum, have been used for B. pertussis detection, although there is limited head-to-head comparison to evaluating the PCR yield from the 3 sampling methods.  Hospitalized infants <6 months of age who fulfilled a broad syndromic criteria of respiratory illness were tested for B. pertussis infection by PCR on paired NPSs and NPAs; or paired NPSs and induced sputum. An exploratory analysis of B. pertussis culture was performed on induced sputum specimens and in a subset of NPSs.  From November 2014 to May 2015, 484 infants with paired NPSs and NPAs were tested; 15 (3.1%) PCR-confirmed pertussis cases were identified, 13 of which were PCR positive on both samples, while 1 each were positive only on NPS or NPA. From March to October 2015, 320 infants had NPSs and induced sputum collected, and 11 (3.4%) pertussis cases were identified by PCR, including 8 (72.7%) positive on both samples, 1 (9.1%) only positive on NPS, and 2 (18.2%) only positive on induced sputum. The 3 types of specimens had similar negative predictive value >99% and sensitivity >83%. Compared to PCR, culture sensitivity was 60% in induced sputum and 40% in NPSs.  Flocked nasopharyngeal swabs, nasopharyngeal aspirates, and induced sputum performed similarly for the detection of B. pertussis infection in young infants by PCR. © The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America.

Comparing the Yield of Nasopharyngeal Swabs, Nasal Aspirates, and Induced Sputum for Detection of Bordetella pertussis in Hospitalized Infants

PubMed Central

Nunes, Marta C.; Soofie, Nasiha; Downs, Sarah; Tebeila, Naume; Mudau, Azwi; de Gouveia, Linda; Madhi, Shabir A.

2016-01-01

Background. Advances in molecular laboratory techniques are changing the landscape of Bordetella pertussis illness diagnosis. Polymerase chain reaction (PCR) assays have greatly improved the sensitivity detection and the turnaround time to diagnosis compared to culture. Moreover, different respiratory specimens, such as flocked nasopharyngeal swabs (NPSs), nasopharyngeal aspirates (NPAs), and induced sputum, have been used for B. pertussis detection, although there is limited head-to-head comparison to evaluating the PCR yield from the 3 sampling methods. Methods. Hospitalized infants <6 months of age who fulfilled a broad syndromic criteria of respiratory illness were tested for B. pertussis infection by PCR on paired NPSs and NPAs; or paired NPSs and induced sputum. An exploratory analysis of B. pertussis culture was performed on induced sputum specimens and in a subset of NPSs. Results. From November 2014 to May 2015, 484 infants with paired NPSs and NPAs were tested; 15 (3.1%) PCR-confirmed pertussis cases were identified, 13 of which were PCR positive on both samples, while 1 each were positive only on NPS or NPA. From March to October 2015, 320 infants had NPSs and induced sputum collected, and 11 (3.4%) pertussis cases were identified by PCR, including 8 (72.7%) positive on both samples, 1 (9.1%) only positive on NPS, and 2 (18.2%) only positive on induced sputum. The 3 types of specimens had similar negative predictive value >99% and sensitivity >83%. Compared to PCR, culture sensitivity was 60% in induced sputum and 40% in NPSs. Conclusions. Flocked nasopharyngeal swabs, nasopharyngeal aspirates, and induced sputum performed similarly for the detection of B. pertussis infection in young infants by PCR. PMID:27838671

[Comparative analysis between diatom nitric acid digestion method and plankton 16S rDNA PCR method].

PubMed

Han, Jun-ge; Wang, Cheng-bao; Li, Xing-biao; Fan, Yan-yan; Feng, Xiang-ping

2013-10-01

To compare and explore the application value of diatom nitric acid digestion method and plankton 16S rDNA PCR method for drowning identification. Forty drowning cases from 2010 to 2011 were collected from Department of Forensic Medicine of Wenzhou Medical University. Samples including lung, kidney, liver and field water from each case were tested with diatom nitric acid digestion method and plankton 16S rDNA PCR method, respectively. The Diatom nitric acid digestion method and plankton 16S rDNA PCR method required 20 g and 2 g of each organ, and 15 mL and 1.5 mL of field water, respectively. The inspection time and detection rate were compared between the two methods. Diatom nitric acid digestion method mainly detected two species of diatoms, Centriae and Pennatae, while plankton 16S rDNA PCR method amplified a length of 162 bp band. The average inspection time of each case of the Diatom nitric acid digestion method was (95.30 +/- 2.78) min less than (325.33 +/- 14.18) min of plankton 16S rDNA PCR method (P < 0.05). The detection rates of two methods for field water and lung were both 100%. For liver and kidney, the detection rate of plankton 16S rDNA PCR method was both 80%, higher than 40% and 30% of diatom nitric acid digestion method (P < 0.05), respectively. The laboratory testing method needs to be appropriately selected according to the specific circumstances in the forensic appraisal of drowning. Compared with diatom nitric acid digestion method, plankton 16S rDNA PCR method has practice values with such advantages as less quantity of samples, huge information and high specificity.

Co-amplification at lower denaturation-temperature PCR combined with unlabled-probe high-resolution melting to detect KRAS codon 12 and 13 mutations in plasma-circulating DNA of pancreatic adenocarcinoma cases.

PubMed

Wu, Jiong; Zhou, Yan; Zhang, Chun-Yan; Song, Bin-Bin; Wang, Bei-Li; Pan, Bai-Shen; Lou, Wen-Hui; Guo, Wei

2014-01-01

The aim of our study was to establish COLD-PCR combined with an unlabeled-probe HRM approach for detecting KRAS codon 12 and 13 mutations in plasma-circulating DNA of pancreatic adenocarcinoma (PA) cases as a novel and effective diagnostic technique. We tested the sensitivity and specificity of this approach with dilutions of known mutated cell lines. We screened 36 plasma-circulating DNA samples, 24 from the disease control group and 25 of a healthy group, to be subsequently sequenced to confirm mutations. Simultaneously, we tested the specimens using conventional PCR followed by HRM and then used target-DNA cloning and sequencing for verification. The ROC and respective AUC were calculated for KRAS mutations and/or serum CA 19-9. It was found that the sensitivity of Sanger reached 0.5% with COLD- PCR, whereas that obtained after conventional PCR did 20%; that of COLD-PCR based on unlabeled-probe HRM, 0.1%. KRAS mutations were identified in 26 of 36 PA cases (72.2%), while none were detected in the disease control and/or healthy group. KRAS mutations were identified both in 26 PA tissues and plasma samples. The AUC of COLD-PCR based unlabeled probe HRM turned out to be 0.861, which when combined with CA 19-9 increased to 0.934. It was concluded that COLD-PCR with unlabeled-probe HRM can be a sensitive and accurate screening technique to detect KRAS codon 12 and 13 mutations in plasma-circulating DNA for diagnosing and treating PA.

Detection of multiple Mycoplasma species in bulk tank milk samples using real-time PCR and conventional culture and comparison of test sensitivities.

PubMed

Justice-Allen, A; Trujillo, J; Goodell, G; Wilson, D

2011-07-01

The objective of this study was to further validate a SYBR PCR protocol for Mycoplasma spp. by comparing it with standard microbial culture in the detection of Mycoplasma spp. in bulk tank milk samples. Additionally, we identified Mycoplasma spp. present by analysis of PCR-generated amplicons [dissociation (melt) temperature (T(m)), length, and DNA sequence]. The research presented herein tests the hypothesis that the SYBR PCR protocol is as sensitive as conventional culture for the detection of Mycoplasma spp. in bulk tank milk samples. Mycoplasmas cause several important disease syndromes in cattle, including mastitis in dairy cows. The standard diagnostic method at the herd level has been microbial isolation of mycoplasmas on 1 of several specialized media and speciation through biochemical or immunological techniques; repeated sampling schemes are recommended. The development of a real-time SYBR PCR protocol offers advantages in decrease of time to detection, cost, and complexity. The T(m) of the double-stranded DNA generated from the PCR reaction was used to detect the presence of and tentatively identify the species of mycoplasmas other than Mycoplasma bovis. In the SYBR PCR protocol, the presence of multiple species of mycoplasmas is indicated by an atypical dissociation curve. Gel electrophoresis and sequencing of the amplicons was used to confirm the mycoplasma species present when a non-M. bovis organism was detected (T(m) not equal to M. bovis) and used to identify all the mycoplasma species present for the samples with atypical dissociation curves. Mycoplasma bovis was identified in 83% of SYBR PCR mycoplasma-positive bulk tank samples. Another mycoplasma was identified either alone or in addition to M. bovis in 25% of SYBR PCR mycoplasma-positive bulk tank milk samples. Four species of mycoplasma other than M. bovis (Mycoplasma alkalescens, Mycoplasma arginini, Mycoplasma bovigenitalium, and Mycoplasma gateae) were identified in bulk tank milk samples tested with this method. Five farms had 2 mycoplasma species occurring at different times in their bulk tanks. Two mycoplasma species were identified in the same bulk tank sample in 7 instances on 2 farms. The finding of multiple Mycoplasma spp. coexisting on a farm and even in the same bulk tank milk sample indicates that the clinical significance of multiple mycoplasma species in the pathology of intramammary infections should be investigated further. In comparison with conventional culture, the SYBR PCR protocol was slightly (but not statistically significantly) more sensitive in the detection of mycoplasmas in bulk tank milk. Copyright © 2011 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

The detection of Yersinia enterocolitica in surface water by quantitative PCR amplification of the ail and yadA genes.

PubMed

Cheyne, Bo M; Van Dyke, Michele I; Anderson, William B; Huck, Peter M

2010-09-01

Yersinia enterocolitica has been detected in surface water, and drinking untreated water is a risk factor for infection. PCR-based methods have been used to detect Y. enterocolitica in various sample types, but quantitative studies have not been conducted in water. In this study, quantitative PCR (qPCR)-based methods targeting the Yersinia virulence genes ail and yadA were used to survey the Grand River watershed in southern Ontario, Canada. Initial testing of reference strains showed that ail and yadA PCR assays were specific for pathogenic biotypes of Y. enterocolitica; however the genes were also detected in one clinical Yersinia intermedia isolate. A survey of surface water from the Grand River watershed showed that both genes were detected at five sampling locations, with the ail and yadA genes detected in 38 and 21% of samples, respectively. Both genes were detected more frequently at colder water temperatures. A screening of Yersinia strains isolated from the watershed showed that the ail gene was detected in three Y. enterocolitica 1A/O:5 isolates. Results of this study show that Yersinia virulence genes were commonly detected in a watershed used as a source of drinking water, and that the occurrence of these genes was seasonal.

[Usefulness of conventional polymerase chain reaction for the detection of Mycoplasma hominis, Ureaplasma spp. and Trichomonas vaginalis in female outpatient's genital samples].

PubMed

Alarcón, Gonzalo; Barraza, Gabriela; Vera, Andrea; Wozniak, Aniela; García, Patricia

2016-02-01

Trichomonas vaginalis, Mycoplasma hominis and Ureaplasma spp. are microorganisms responsible for genitourinary and pregnancy pathologies. Nucleic acid amplification methods have shown several advantages, but have not been widely studied for the detection of these microorganisms. To implement a conventional polymerase chain reaction (PCR) for the detection of the microorganisms and to compare its results versus the methods currently used at our laboratory. 91 available samples were processed by PCR, culture (M. hominis y Ureaplasma spp.) and wet mount (T vaginalis). Results were compared and statistically analyzed by kappa agreement test. 85, 80 and 87 samples resulted in agreement for the detection of M. hominis, Ureaplasma spp. y T. vaginalis, respectively. For M. hominis and Ureaplasma spp., agreement was substantial, whereas for T. vaginalis it was moderate, however, for the latter, PCR detected more cases than wet mount. We recommend the implementation of PCR for detection of T. vaginalis whereas culture kit is still a useful method for the other microorganisms.

Effect of Sequence Polymorphisms on Performance of Two Real-Time PCR Assays for Detection of Herpes Simplex Virus

PubMed Central

Stevenson, Jeffery; Hymas, Weston; Hillyard, David

2005-01-01

Herpes simplex virus (HSV) is the most common cause of acquired, sporadic encephalitis in the United States. PCR identification of HSV in spinal fluid has become the diagnostic gold standard due to its sensitivity and potential for speed, replacing other methods such as culture. We developed a real-time PCR assay to detect HSV, using a new type of hybridization probe, the Eclipse probe. In this study, we ran 323 samples (171 positives and 152 negatives) with the Eclipse real-time PCR assay and compared these results with another PCR assay using gel detection. The real-time assay agreed with our reference method for 319 out of the 323 samples tested (99%). Using two different real-time PCR platforms, we discovered that SNPs within the amplicon's probe binding region that are used to distinguish HSV-1 from HSV-2 can decrease assay sensitivity. This problem is potentially a general one for assays using fluorescent probes to detect target amplification in a real-time format. While real-time PCR can be a powerful tool in the field of infectious disease, careful sequence evaluation and clinical validation are essential in creating an effective assay. PMID:15872272

Real-Time Polymerase Chain Reaction Detection of Angiostrongylus cantonensis DNA in Cerebrospinal Fluid from Patients with Eosinophilic Meningitis

PubMed Central

Qvarnstrom, Yvonne; Xayavong, Maniphet; da Silva, Ana Cristina Aramburu; Park, Sarah Y.; Whelen, A. Christian; Calimlim, Precilia S.; Sciulli, Rebecca H.; Honda, Stacey A. A.; Higa, Karen; Kitsutani, Paul; Chea, Nora; Heng, Seng; Johnson, Stuart; Graeff-Teixeira, Carlos; Fox, LeAnne M.; da Silva, Alexandre J.

2016-01-01

Angiostrongylus cantonensis is the most common infectious cause of eosinophilic meningitis. Timely diagnosis of these infections is difficult, partly because reliable laboratory diagnostic methods are unavailable. The aim of this study was to evaluate the usefulness of a real-time polymerase chain reaction (PCR) assay for the detection of A. cantonensis DNA in human cerebrospinal fluid (CSF) specimens. A total of 49 CSF specimens from 33 patients with eosinophilic meningitis were included: A. cantonensis DNA was detected in 32 CSF specimens, from 22 patients. Four patients had intermittently positive and negative real-time PCR results on subsequent samples, indicating that the level of A. cantonensis DNA present in CSF may fluctuate during the course of the illness. Immunodiagnosis and/or supplemental PCR testing supported the real-time PCR findings for 30 patients. On the basis of these observations, this real-time PCR assay can be useful to detect A. cantonensis in the CSF from patients with eosinophilic meningitis. PMID:26526920

Standardization of a TaqMan-based real-time PCR for the detection of Mycobacterium tuberculosis-complex in human sputum.

PubMed

Barletta, Francesca; Vandelannoote, Koen; Collantes, Jimena; Evans, Carlton A; Arévalo, Jorge; Rigouts, Leen

2014-10-01

Real-time polymerase chain reaction (qPCR) was optimized for detecting Mycobacterium tuberculosis in sputum. Sputum was collected from patients (N = 112) with suspected pulmonary tuberculosis, tested by smear microscopy, decontaminated, and split into equal aliquots that were cultured in Löwenstein-Jensen medium and tested by qPCR for the small mobile genetic element IS6110. The human ERV3 sequence was used as an internal control. 3 of 112 (3%) qPCR failed. For the remaining 109 samples, qPCR diagnosed tuberculosis in 79 of 84 patients with culture-proven tuberculosis, and sensitivity was greater than microscopy (94% versus 76%, respectively, P < 0.05). The qPCR sensitivity was similar (P = 0.9) for smear-positive (94%, 60 of 64) and smear-negative (95%, 19 of 20) samples. The qPCR was negative for 24 of 25 of the sputa with negative microscopy and culture (diagnostic specificity 96%). The qPCR had 99.5% sensitivity and specificity for 211 quality control samples including 84 non-tuberculosis mycobacteria. The qPCR cost ∼5US$ per sample and provided same-day results compared with 2-6 weeks for culture. © The American Society of Tropical Medicine and Hygiene.

Evaluation of a Campylobacter fetus subspecies venerealis real-time quantitative polymerase chain reaction for direct analysis of bovine preputial samples

PubMed Central

Chaban, Bonnie; Chu, Shirley; Hendrick, Steven; Waldner, Cheryl; Hill, Janet E.

2012-01-01

The detection and subspeciation of Campylobacter fetus subsp. venerealis (CFV) from veterinary samples is important for both clinical and economic reasons. Campylobacter fetus subsp. venerealis is the causative agent of bovine genital campylobacteriosis, a venereal disease that can lead to serious reproductive problems in cattle, and strict international regulations require animals and animal products to be CFV-free for trade. This study evaluated methods reported in the literature for CFV detection and reports the translation of an extensively tested CFV-specific polymerase chain reaction (PCR) primer set; including the VenSF/VenSR primers and a real-time, quantitative PCR (qPCR) platform using SYBR Green chemistry. Three methods of preputial sample preparation for direct qPCR were evaluated and a heat lysis DNA extraction method was shown to allow for CFV detection at the level of approximately one cell equivalent per reaction (or 1.0 × 103 CFU/mL) from prepuce. The optimized sample preparation and qPCR protocols were then used to evaluate 3 western Canadian bull cohorts, which included 377 bulls, for CFV. The qPCR assay detected 11 positive bulls for the CFV-specific parA gene target. DNA sequence data confirmed the identity of the amplified product and revealed that positive samples were comprised of 2 sequence types; one identical to previously reported CFV parA gene sequences and one with a 9% sequence divergence. These results add valuable information towards our understanding of an important CFV subspeciation target and offer a significantly improved format for an internationally recognized PCR test. PMID:23277694

Analytical and Clinical Performance of the CDC Real Time RT-PCR Assay for Detection and Typing of Dengue Virus

PubMed Central

Santiago, Gilberto A.; Vergne, Edgardo; Quiles, Yashira; Cosme, Joan; Vazquez, Jesus; Medina, Juan F.; Medina, Freddy; Colón, Candimar; Margolis, Harold; Muñoz-Jordán, Jorge L.

2013-01-01

Dengue is an acute illness caused by the positive-strand RNA dengue virus (DENV). There are four genetically distinct DENVs (DENV-1–4) that cause disease in tropical and subtropical countries. Most patients are viremic when they present with symptoms; therefore, RT-PCR has been increasingly used in dengue diagnosis. The CDC DENV-1–4 RT-PCR Assay has been developed as an in-vitro diagnostic platform and was recently approved by the US Food and Drug Administration (FDA) for detection of dengue in patients with signs or symptoms of mild or severe dengue. The primers and probes of this test have been designed to detect currently circulating strains of DENV-1–4 from around the world at comparable sensitivity. In a retrospective study with 102 dengue cases confirmed by IgM anti-DENV seroconversion in the convalescent sample, the RT-PCR Assay detected DENV RNA in 98.04% of the paired acute samples. Using sequencing as a positive indicator, the RT-PCR Assay had a 97.92% positive agreement in 86 suspected dengue patients with a single acute serum sample. After extensive validations, the RT-PCR Assay performance was highly reproducible when evaluated across three independent testing sites, did not produce false positive results for etiologic agents of other febrile illnesses, and was not affected by pathological levels of potentially interfering biomolecules. These results indicate that the CDC DENV-1–4 RT-PCR Assay provides a reliable diagnostic platform capable for confirming dengue in suspected cases. PMID:23875046

Evaluation of urine for Leishmania infantum DNA detection by real-time quantitative PCR.

PubMed

Pessoa-E-Silva, Rômulo; Mendonça Trajano-Silva, Lays Adrianne; Lopes da Silva, Maria Almerice; da Cunha Gonçalves-de-Albuquerque, Suênia; de Goes, Tayná Correia; Silva de Morais, Rayana Carla; Lopes de Melo, Fábio; de Paiva-Cavalcanti, Milena

2016-12-01

The availability of some sorts of biological samples which require noninvasive collection methods has led to an even greater interest in applying molecular biology on visceral leishmaniasis (VL) diagnosis, since these samples increase the safety and comfort of both patients and health professionals. In this context, this work aimed to evaluate the suitability of the urine as a specimen for Leishmania infantum kinetoplast DNA detection by real-time quantitative PCR (qPCR). Subsequent to the reproducibility analysis, the detection limit of the qPCR assay was set at 5fg (~0.025 parasites) per μL of urine. From the comparative analysis performed with a set of diagnostic criteria (serological and molecular reference tests), concordance value of 96.08% was obtained (VL-suspected and HIV/AIDS patients, n=51) (P>0.05). Kappa coefficient (95% CI) indicated a good agreement between the test and the set of diagnostic criteria (k=0.778±0.151). The detection of Leishmania DNA in urine by qPCR was possible in untreated individuals, and in those with or without suggestive renal impairment. Fast depletion of the parasite's DNA in urine after treatment (from one dose of meglumine antimoniate) was suggested by negative qPCR results, thus indicating it as a potential alternative specimen to follow up the efficacy of therapeutic approaches. Even when evaluated in a clinically heterogeneous set of patients, the urine showed good prospect as sample for VL diagnosis by qPCR, also indicating a good negative predictive value for untreated suspected patients. Copyright © 2016 Elsevier B.V. All rights reserved.

Development of two real-time polymerase chain reaction assays to detect Actinobacillus pleuropneumoniae serovars 1-9-11 and serovar 2.

PubMed

Marois-Créhan, Corinne; Lacouture, Sonia; Jacques, Mario; Fittipaldi, Nahuel; Kobisch, Marylène; Gottschalk, Marcelo

2014-01-01

Two real-time, or quantitative, polymerase chain reaction (qPCR) assays were developed to detect Actinobacillus pleuropneumoniae serovars 1-9-11 (highly related serovars with similar virulence potential) and serovar 2, respectively. The specificity of these assays was verified on a collection of 294 strains, which included all 16 reference A. pleuropneumoniae strains (including serovars 5a and 5b), 263 A. pleuropneumoniae field strains isolated between 1992 and 2009 in different countries, and 15 bacterial strains other than A. pleuropneumoniae. The detection levels of both qPCR tests were evaluated using 10-fold dilutions of chromosomal DNA from reference strains of A. pleuropneumoniae serovars 1 and 2, and the detection limit for both assays was 50 fg per assay. The analytical sensitivities of the qPCR tests were also estimated by using pure cultures and tonsils experimentally spiked with A. pleuropneumoniae. The detection threshold was 2.5 × 10(4) colony forming units (CFU)/ml and 2.9 × 10(5) CFU/0.1 g of tonsil, respectively, for both assays. These specific and sensitive tests can be used for the serotyping of A. pleuropneumoniae in diagnostic laboratories to control porcine pleuropneumonia.

Diagnosis of Helicobacter pylori: What should be the gold standard?

PubMed Central

Patel, Saurabh Kumar; Pratap, Chandra Bhan; Jain, Ashok Kumar; Gulati, Anil Kumar; Nath, Gopal

2014-01-01

Since the discovery of Helicobacter pylori (H. pylori) in 1983, numerous detection methods for the presence of the bacterium have been developed. Each one of them has been associated with advantages and disadvantages. Noninvasive tests such as serology, 13C urea breath test (UBT) and stool antigen tests are usually preferred by the clinicians. Serology has its own limitation especially in endemic areas while 13C UBT is technically very demanding. The stool antigen detection method, although specific, is usually associated with poor sensitivity. The 13C UBT is believed to be specific, but with present revelation of the fact that stomach is colonized by many other urease producing bacteria makes it questionable. Histology, culture, rapid urease test and polymerase chain reaction (PCR) are the tests which are carried out on antral biopsies collected by invasive means. Histology has been proposed to be very sensitive and specific but the question is how by simply looking the morphology of the bacteria in the microscope, one can claim that the curved bacterium is exclusively H. pylori. Rapid urease test (RUT), the doctor’s test, is also challenged because the presence of other urease producing bacteria in the stomach cannot be denied. Moreover, RUT has been reported with poor sensitivity specially, when density of the bacterium is low. Isolation of H. pylori is essential to investigate its growth requirements, antibiotic susceptibility testing, studying virulence factor to develop vaccine and many more explorations. It has also got several disadvantages i.e., special condition for transporting, media, incubation and few days waiting for the colonies to appear, apart from the speed essentially needed to process the specimens. Till date, majority of the microbiological laboratories in the world are not equipped and trained to isolate such fastidious bacterium. The option left is PCR methods to detect H. pylori’s DNA in gastric mucosa, gastric juice, saliva, dental plaques and environmental specimens. There are speculations for false positivity due to detection of non-pylori Helicobacters due to genetic sharing; and false negativity due to low bacterial counts and presence of PCR inhibitors. However, specimen collection, transportation and processing do not require speed and special conditions. PCR based diagnosis may be considered as gold standard by designing primers extremely specific to H. pylori and targeting at least more than one conserved genes. Similarly specificity of PCR may be improved by use of internal Primers. Further, nested PCR will take care of false negatives by countering the effect of PCR inhibitors and low bacterial counts. Therefore, nested PCR based methods if performed properly, may be proposed as gold standard test. PMID:25278682

Direct PCR - A rapid method for multiplexed detection of different serotypes of Salmonella in enriched pork meat samples.

PubMed

Chin, Wai Hoe; Sun, Yi; Høgberg, Jonas; Quyen, Than Linh; Engelsmann, Pia; Wolff, Anders; Bang, Dang Duong

2017-04-01

Salmonellosis, an infectious disease caused by Salmonella spp., is one of the most common foodborne diseases. Isolation and identification of Salmonella by conventional bacterial culture method is time consuming. In response to the demand for rapid on line or at site detection of pathogens, in this study, we developed a multiplex Direct PCR method for rapid detection of different Salmonella serotypes directly from pork meat samples without any DNA purification steps. An inhibitor-resistant Phusion Pfu DNA polymerase was used to overcome PCR inhibition. Four pairs of primers including a pair of newly designed primers targeting Salmonella spp. at subtype level were incorporated in the multiplex Direct PCR. To maximize the efficiency of the Direct PCR, the ratio between sample and dilution buffer was optimized. The sensitivity and specificity of the multiplex Direct PCR were tested using naturally contaminated pork meat samples for detecting and subtyping of Salmonella spp. Conventional bacterial culture methods were used as reference to evaluate the performance of the multiplex Direct PCR. Relative accuracy, sensitivity and specificity of 98.8%; 97.6% and 100%, respectively, were achieved by the method. Application of the multiplex Direct PCR to detect Salmonella in pork meat at slaughter reduces the time of detection from 5 to 6 days by conventional bacterial culture and serotyping methods to 14 h (including 12 h enrichment time). Furthermore, the method poses a possibility of miniaturization and integration into a point-of-need Lab-on-a-chip system for rapid online pathogen detection. Copyright © 2016 Elsevier Ltd. All rights reserved.

Multiplex picodroplet digital PCR to detect KRAS mutations in circulating DNA from the plasma of colorectal cancer patients.

PubMed

Taly, Valerie; Pekin, Deniz; Benhaim, Leonor; Kotsopoulos, Steve K; Le Corre, Delphine; Li, Xinyu; Atochin, Ivan; Link, Darren R; Griffiths, Andrew D; Pallier, Karine; Blons, Hélène; Bouché, Olivier; Landi, Bruno; Hutchison, J Brian; Laurent-Puig, Pierre

2013-12-01

Multiplex digital PCR (dPCR) enables noninvasive and sensitive detection of circulating tumor DNA with performance unachievable by current molecular-detection approaches. Furthermore, picodroplet dPCR facilitates simultaneous screening for multiple mutations from the same sample. We investigated the utility of multiplex dPCR to screen for the 7 most common mutations in codons 12 and 13 of the KRAS (Kirsten rat sarcoma viral oncogene homolog) oncogene from plasma samples of patients with metastatic colorectal cancer. Fifty plasma samples were tested from patients for whom the primary tumor biopsy tissue DNA had been characterized by quantitative PCR. Tumor characterization revealed that 19 patient tumors had KRAS mutations. Multiplex dPCR analysis of the plasma DNA prepared from these samples identified 14 samples that matched the mutation identified in the tumor, 1 sample contained a different KRAS mutation, and 4 samples had no detectable mutation. Among the tumor samples that were wild type for KRAS, 2 KRAS mutations were identified in the corresponding plasma samples. Duplex dPCR (i.e., wild-type and single-mutation assay) was also used to analyze plasma samples from patients with KRAS-mutated tumors and 5 samples expected to contain the BRAF (v-raf murine sarcoma viral oncogene homolog B) V600E mutation. The results for the duplex analysis matched those for the multiplex analysis for KRAS-mutated samples and, owing to its higher sensitivity, enabled detection of 2 additional samples with low levels of KRAS-mutated DNA. All 5 samples with BRAF mutations were detected. This work demonstrates the clinical utility of multiplex dPCR to screen for multiple mutations simultaneously with a sensitivity sufficient to detect mutations in circulating DNA obtained by noninvasive blood collection.

Rapid and sensitive detection of Feline immunodeficiency virus using an insulated isothermal PCR-based assay with a point-of-need PCR detection platform.

PubMed

Wilkes, Rebecca Penrose; Kania, Stephen A; Tsai, Yun-Long; Lee, Pei-Yu Alison; Chang, Hsiu-Hui; Ma, Li-Juan; Chang, Hsiao-Fen Grace; Wang, Hwa-Tang Thomas

2015-07-01

Feline immunodeficiency virus (FIV) is an important infectious agent of cats. Clinical syndromes resulting from FIV infection include immunodeficiency, opportunistic infections, and neoplasia. In our study, a 5' long terminal repeat/gag region-based reverse transcription insulated isothermal polymerase chain reaction (RT-iiPCR) was developed to amplify all known FIV strains to facilitate point-of-need FIV diagnosis. The RT-iiPCR method was applied in a point-of-need PCR detection platform--a field-deployable device capable of generating automatically interpreted RT-iiPCR results from nucleic acids within 1 hr. Limit of detection 95% of FIV RT-iiPCR was calculated to be 95 copies standard in vitro transcription RNA per reaction. Endpoint dilution studies with serial dilutions of an ATCC FIV type strain showed that the sensitivity of lyophilized FIV RT-iiPCR reagent was comparable to that of a reference nested PCR. The established reaction did not amplify any nontargeted feline pathogens, including Felid herpesvirus 1, feline coronavirus, Feline calicivirus, Feline leukemia virus, Mycoplasma haemofelis, and Chlamydophila felis. Based on analysis of 76 clinical samples (including blood and bone marrow) with the FIV RT-iiPCR, test sensitivity was 97.78% (44/45), specificity was 100.00% (31/31), and agreement was 98.65% (75/76), determined against a reference nested-PCR assay. A kappa value of 0.97 indicated excellent correlation between these 2 methods. The lyophilized FIV RT-iiPCR reagent, deployed on a user-friendly portable device, has potential utility for rapid and easy point-of-need detection of FIV in cats. © 2015 The Author(s).

Broad-Range PCR Coupled with Electrospray Ionization Time of Flight Mass Spectrometry for Detection of Bacteremia and Fungemia in Patients with Neutropenic Fever

PubMed Central

Maertens, J.; Bueselinck, K.; Lagrou, K.

2016-01-01

Infection is an important complication in patients with hematologic malignancies or solid tumors undergoing intensive cytotoxic chemotherapy. In only 20 to 30% of the febrile neutropenic episodes, an infectious agent is detected by conventional cultures. In this prospective study, the performance of broad-range PCR coupled with electrospray ionization time of flight mass spectrometry (PCR/ESI-MS) technology was compared to conventional blood cultures (BC) in a consecutive series of samples from high-risk hematology patients. In 74 patients, BC and a whole-blood sample for PCR/ESI-MS (Iridica BAC BSI; Abbott, Carlsbad, CA, USA) were collected at the start of each febrile neutropenic episode and, in case of persistent fever, also at day 5. During 100 different febrile episodes, 105 blood samples were collected and analyzed by PCR/ESI-MS. There was evidence of a bloodstream infection (BSI) in 36/105 cases (34%), based on 14 cases with both PCR/ESI-MS and BC positivity, 17 cases with BC positivity only, and 5 cases with PCR/ESI-MS positivity only. The sensitivity of PCR/ESI-MS was 45%, specificity was 93%, and the negative predictive value was 80% compared to blood culture. PCR/ESI-MS detected definite pathogens (Fusobacterium nucleatum and Streptococcus pneumoniae) missed by BC, whereas it missed both Gram-negative and Gram-positive organisms detected by BC. PCR/ESI-MS testing detected additional microorganisms but showed a low sensitivity (45%) compared to BC in neutropenic patients. Our results indicate a lower concordance between BC and PCR/ESI-MS in the neutropenic population than what has been previously reported in other patient groups with normal white blood cell distribution, and a lower sensitivity than other PCR-based methods. PMID:27440820

Evaluation of Targeted Next-Generation Sequencing for Detection of Bovine Pathogens in Clinical Samples.

PubMed

Anis, Eman; Hawkins, Ian K; Ilha, Marcia R S; Woldemeskel, Moges W; Saliki, Jeremiah T; Wilkes, Rebecca P

2018-07-01

The laboratory diagnosis of infectious diseases, especially those caused by mixed infections, is challenging. Routinely, it requires submission of multiple samples to separate laboratories. Advances in next-generation sequencing (NGS) have provided the opportunity for development of a comprehensive method to identify infectious agents. This study describes the use of target-specific primers for PCR-mediated amplification with the NGS technology in which pathogen genomic regions of interest are enriched and selectively sequenced from clinical samples. In the study, 198 primers were designed to target 43 common bovine and small-ruminant bacterial, fungal, viral, and parasitic pathogens, and a bioinformatics tool was specifically constructed for the detection of targeted pathogens. The primers were confirmed to detect the intended pathogens by testing reference strains and isolates. The method was then validated using 60 clinical samples (including tissues, feces, and milk) that were also tested with other routine diagnostic techniques. The detection limits of the targeted NGS method were evaluated using 10 representative pathogens that were also tested by quantitative PCR (qPCR), and the NGS method was able to detect the organisms from samples with qPCR threshold cycle ( C T ) values in the 30s. The method was successful for the detection of multiple pathogens in the clinical samples, including some additional pathogens missed by the routine techniques because the specific tests needed for the particular organisms were not performed. The results demonstrate the feasibility of the approach and indicate that it is possible to incorporate NGS as a diagnostic tool in a cost-effective manner into a veterinary diagnostic laboratory. Copyright © 2018 Anis et al.

Comparison of diagnostic techniques for the detection of Cryptosporidium oocysts in animal samples

PubMed Central

Mirhashemi, Marzieh Ezzaty; Zintl, Annetta; Grant, Tim; Lucy, Frances E.; Mulcahy, Grace; De Waal, Theo

2015-01-01

While a large number of laboratory methods for the detection of Cryptosporidium oocysts in faecal samples are now available, their efficacy for identifying asymptomatic cases of cryptosporidiosis is poorly understood. This study was carried out to determine a reliable screening test for epidemiological studies in livestock. In addition, three molecular tests were compared to identify Cryptosporidium species responsible for the infection in cattle, sheep and horses. A variety of diagnostic tests including microscopic (Kinyoun's staining), immunological (Direct Fluorescence Antibody tests or DFAT), enzyme-linked immunosorbent assay (ELISA), and molecular methods (nested PCR) were compared to assess their ability to detect Cryptosporidium in cattle, horse and sheep faecal samples. The results indicate that the sensitivity and specificity of each test is highly dependent on the input samples; while Kinyoun's and DFAT proved to be reliable screening tools for cattle samples, DFAT and PCR analysis (targeted at the 18S rRNA gene fragment) were more sensitive for screening sheep and horse samples. Finally different PCR primer sets targeted at the same region resulted in the preferential amplification of certain Cryptosporidium species when multiple species were present in the sample. Therefore, for identification of Cryptosporidium spp. in the event of asymptomatic cryptosporidiosis, the combination of different 18S rRNA nested PCR primer sets is recommended for further epidemiological applications and also tracking the sources of infection. PMID:25662435

Clinical Evaluation of a Loop-Mediated Amplification Kit for Diagnosis of Imported Malaria

PubMed Central

Polley, Spencer D.; González, Iveth J.; Mohamed, Deqa; Daly, Rosemarie; Bowers, Kathy; Watson, Julie; Mewse, Emma; Armstrong, Margaret; Gray, Christen; Perkins, Mark D.; Bell, David; Kanda, Hidetoshi; Tomita, Norihiro; Kubota, Yutaka; Mori, Yasuyoshi; Chiodini, Peter L.; Sutherland, Colin J.

2013-01-01

Background. Diagnosis of malaria relies on parasite detection by microscopy or antigen detection; both fail to detect low-density infections. New tests providing rapid, sensitive diagnosis with minimal need for training would enhance both malaria diagnosis and malaria control activities. We determined the diagnostic accuracy of a new loop-mediated amplification (LAMP) kit in febrile returned travelers. Methods. The kit was evaluated in sequential blood samples from returned travelers sent for pathogen testing to a specialist parasitology laboratory. Microscopy was performed, and then malaria LAMP was performed using Plasmodium genus and Plasmodium falciparum–specific tests in parallel. Nested polymerase chain reaction (PCR) was performed on all samples as the reference standard. Primary outcome measures for diagnostic accuracy were sensitivity and specificity of LAMP results, compared with those of nested PCR. Results. A total of 705 samples were tested in the primary analysis. Sensitivity and specificity were 98.4% and 98.1%, respectively, for the LAMP P. falciparum primers and 97.0% and 99.2%, respectively, for the Plasmodium genus primers. Post hoc repeat PCR analysis of all 15 tests with discrepant results resolved 4 results in favor of LAMP, suggesting that the primary analysis had underestimated diagnostic accuracy. Conclusions. Malaria LAMP had a diagnostic accuracy similar to that of nested PCR, with a greatly reduced time to result, and was superior to expert microscopy. PMID:23633403

Comparison of three human papillomavirus DNA detection methods: Next generation sequencing, multiplex-PCR and nested-PCR followed by Sanger based sequencing.

PubMed

da Fonseca, Allex Jardim; Galvão, Renata Silva; Miranda, Angelica Espinosa; Ferreira, Luiz Carlos de Lima; Chen, Zigui

2016-05-01

To compare the diagnostic performance for HPV infection using three laboratorial techniques. Ninty-five cervicovaginal samples were randomly selected; each was tested for HPV DNA and genotypes using 3 methods in parallel: Multiplex-PCR, the Nested PCR followed by Sanger sequencing, and the Next_Gen Sequencing (NGS) with two assays (NGS-A1, NGS-A2). The study was approved by the Brazilian National IRB (CONEP protocol 16,800). The prevalence of HPV by the NGS assays was higher than that using the Multiplex-PCR (64.2% vs. 45.2%, respectively; P = 0.001) and the Nested-PCR (64.2% vs. 49.5%, respectively; P = 0.003). NGS also showed better performance in detecting high-risk HPV (HR-HPV) and HPV16. There was a weak interobservers agreement between the results of Multiplex-PCR and Nested-PCR in relation to NGS for the diagnosis of HPV infection, and a moderate correlation for HR-HPV detection. Both NGS assays showed a strong correlation for detection of HPVs (k = 0.86), HR-HPVs (k = 0.91), HPV16 (k = 0.92) and HPV18 (k = 0.91). NGS is more sensitive than the traditional Sanger sequencing and the Multiplex PCR to genotype HPVs, with promising ability to detect multiple infections, and may have the potential to establish an alternative method for the diagnosis and genotyping of HPV. © 2015 Wiley Periodicals, Inc.

Identification of Mycobacterium tuberculosis in Clinical Specimens of Patients Suspected of Having Extrapulmonary Tuberculosis by Application of Nested PCR on Five Different Genes

PubMed Central

Khosravi, Azar D.; Alami, Ameneh; Meghdadi, Hossein; Hosseini, Atta A.

2017-01-01

Definitive and rapid diagnosis of extrapulmonary tuberculosis (EPTB) is challenging since conventional techniques have limitations due to the paucibacillary nature of the disease. To increase the sensitivity of detection of Mycobacterium tuberculosis (MTB) in EPTB specimens, we performed a nested PCR assay targeting several genes of MTB on EPTB specimens. A total of 100 clinical specimens from suspected cases of EPTB were processed. Standard staining for acid fast bacilli (AFB) was performed as the preliminary screening test. Extracted DNAs from specimens were subjected to Nested PCR technique for the detection of five different MTB target genes of IS6110, IS1081, hsp65kd, mbp64, and mtp40. On performing AFB staining, only 13% of specimens were positive, of which ascites fluid (33.3%), followed by pleural effusion (30.8%) showed the greatest AFB positivity rate. We demonstrated slight improvement in yields in lymph node which comprised the majority of specimens in this study, by employing PCR targeted to IS6110- and hsp65-genes in comparison to AFB staining. However, the yields in ascites fluid and pleural effusion were not substantially improved by PCR, but those from bone and wound were, as in nested PCR employing either gene, the same positivity rate were obtained for ascites fluid (33.3%), while for pleural effusion specimens only IS1081 based PCR showed identical positivity rate with AFB stain (30.8%). The results for bone and wound specimens, however, demonstrated an improved yield mainly by employing IS1081 gene. Here, we report higher detection rate of EPTB in clinical specimens using five different targeted MTB genes. This nested PCR approach facilitates the comparison and the selection of the most frequently detected genes. Of course this study demonstrated the priority of IS1081 followed by mtp40 and IS6110, among the five tested genes and indicates the effectiveness of any of the three genes in the design of an efficient nested-PCR test that facilitates an early diagnosis of paucibacillary EPTB cases, which are difficult to diagnose with the available standard. PMID:28144587

Identification of Mycobacterium tuberculosis in Clinical Specimens of Patients Suspected of Having Extrapulmonary Tuberculosis by Application of Nested PCR on Five Different Genes.

PubMed

Khosravi, Azar D; Alami, Ameneh; Meghdadi, Hossein; Hosseini, Atta A

2017-01-01

Definitive and rapid diagnosis of extrapulmonary tuberculosis (EPTB) is challenging since conventional techniques have limitations due to the paucibacillary nature of the disease. To increase the sensitivity of detection of Mycobacterium tuberculosis (MTB) in EPTB specimens, we performed a nested PCR assay targeting several genes of MTB on EPTB specimens. A total of 100 clinical specimens from suspected cases of EPTB were processed. Standard staining for acid fast bacilli (AFB) was performed as the preliminary screening test. Extracted DNAs from specimens were subjected to Nested PCR technique for the detection of five different MTB target genes of IS6110, IS1081, hsp65kd, mbp64 , and mtp40 . On performing AFB staining, only 13% of specimens were positive, of which ascites fluid (33.3%), followed by pleural effusion (30.8%) showed the greatest AFB positivity rate. We demonstrated slight improvement in yields in lymph node which comprised the majority of specimens in this study, by employing PCR targeted to IS6110 - and hsp65-genes in comparison to AFB staining. However, the yields in ascites fluid and pleural effusion were not substantially improved by PCR, but those from bone and wound were, as in nested PCR employing either gene, the same positivity rate were obtained for ascites fluid (33.3%), while for pleural effusion specimens only IS1081 based PCR showed identical positivity rate with AFB stain (30.8%). The results for bone and wound specimens, however, demonstrated an improved yield mainly by employing IS1081 gene. Here, we report higher detection rate of EPTB in clinical specimens using five different targeted MTB genes. This nested PCR approach facilitates the comparison and the selection of the most frequently detected genes. Of course this study demonstrated the priority of IS1081 followed by mtp40 and IS6110 , among the five tested genes and indicates the effectiveness of any of the three genes in the design of an efficient nested-PCR test that facilitates an early diagnosis of paucibacillary EPTB cases, which are difficult to diagnose with the available standard.

Evaluation by latent class analysis of a magnetic capture based DNA extraction followed by real-time qPCR as a new diagnostic method for detection of Echinococcus multilocularis in definitive hosts.

PubMed

Maas, Miriam; van Roon, Annika; Dam-Deisz, Cecile; Opsteegh, Marieke; Massolo, Alessandro; Deksne, Gunita; Teunis, Peter; van der Giessen, Joke

2016-10-30

A new method, based on a magnetic capture based DNA extraction followed by qPCR, was developed for the detection of the zoonotic parasite Echinococcus multilocularis in definitive hosts. Latent class analysis was used to compare this new method with the currently used phenol-chloroform DNA extraction followed by single tube nested PCR. In total, 60 red foxes and coyotes from three different locations were tested with both molecular methods and the sedimentation and counting technique (SCT) or intestinal scraping technique (IST). Though based on a limited number of samples, it could be established that the magnetic capture based DNA extraction followed by qPCR showed similar sensitivity and specificity as the currently used phenol-chloroform DNA extraction followed by single tube nested PCR. All methods have a high specificity as shown by Bayesian latent class analysis. Both molecular assays have higher sensitivities than the combined SCT and IST, though the uncertainties in sensitivity estimates were wide for all assays tested. The magnetic capture based DNA extraction followed by qPCR has the advantage of not requiring hazardous chemicals like the phenol-chloroform DNA extraction followed by single tube nested PCR. This supports the replacement of the phenol-chloroform DNA extraction followed by single tube nested PCR by the magnetic capture based DNA extraction followed by qPCR for molecular detection of E. multilocularis in definitive hosts. Copyright © 2016 Elsevier B.V. All rights reserved.

Evaluation of a combined triple method to detect causative HPV in oral and oropharyngeal squamous cell carcinomas: p16 Immunohistochemistry, Consensus PCR HPV-DNA, and In Situ Hybridization

PubMed Central

2012-01-01

Background Recent emerging evidences identify Human Papillomavirus (HPV) related Head and Neck squamous cell carcinomas (HN-SCCs) as a separate subgroup among Head and Neck Cancers with different epidemiology, histopathological characteristics, therapeutic response to chemo-radiation treatment and clinical outcome. However, there is not a worldwide consensus on the methods to be used in clinical practice. The endpoint of this study was to demonstrate the reliability of a triple method which combines evaluation of: 1. p16 protein expression by immunohistochemistry (p16-IHC); 2. HPV-DNA genotyping by consensus HPV-DNA PCR methods (Consensus PCR); and 3 viral integration into the host by in situ hybridization method (ISH). This triple method has been applied to HN-SCC originated from oral cavity (OSCC) and oropharynx (OPSCC), the two anatomical sites in which high risk (HR) HPVs have been clearly implicated as etiologic factors. Methylation-Specific PCR (MSP) was performed to study inactivation of p16-CDKN2a locus by epigenetic events. Reliability of multiple methods was measured by Kappa statistics. Results All the HN-SCCs confirmed HPV positive by PCR and/or ISH were also p16 positive by IHC, with the latter showing a very high level of sensitivity as single test (100% in both OSCC and OPSCC) but lower specificity level (74% in OSCC and 93% in OPSCC). Concordance analysis between ISH and Consensus PCR showed a faint agreement in OPSCC (κ = 0.38) and a moderate agreement in OSCC (κ = 0.44). Furthermore, the addition of double positive score (ISHpositive and Consensus PCR positive) increased significantly the specificity of HR-HPV detection on formalin-fixed paraffin embedded (FFPE) samples (100% in OSCC and 78.5% in OPSCC), but reduced the sensitivity (33% in OSCC and 60% in OPSCC). The significant reduction of sensitivity by the double method was compensated by a very high sensitivity of p16-IHC detection in the triple approach. Conclusions Although HR-HPVs detection is of utmost importance in clinical settings for the Head and Neck Cancer patients, there is no consensus on which to consider the 'golden standard' among the numerous detection methods available either as single test or combinations. Until recently, quantitative E6 RNA PCR has been considered the 'golden standard' since it was demonstrated to have very high accuracy level and very high statistical significance associated with prognostic parameters. In contrast, quantitative E6 DNA PCR has proven to have very high level of accuracy but lesser prognostic association with clinical outcome than the HPV E6 oncoprotein RNA PCR. However, although it is theoretically possible to perform quantitative PCR detection methods also on FFPE samples, they reach the maximum of accuracy on fresh frozen tissue. Furthermore, worldwide diagnostic laboratories have not all the same ability to analyze simultaneously both FFPE and fresh tissues with these quantitative molecular detection methods. Therefore, in the current clinical practice a p16-IHC test is considered as sufficient for HPV diagnostic in accordance with the recently published Head and Neck Cancer international guidelines. Although p16-IHC may serve as a good prognostic indicator, our study clearly demonstrated that it is not satisfactory when used exclusively as the only HPV detecting method. Adding ISH, although known as less sensitive than PCR-based detection methods, has the advantage to preserve the morphological context of HPV-DNA signals in FFPE samples and, thus increase the overall specificity of p16/Consensus PCR combination tests. PMID:22376902

Rapid Detection of the Chlamydiaceae and Other Families in the Order Chlamydiales: Three PCR Tests

PubMed Central

Everett, Karin D. E.; Hornung, Linda J.; Andersen, Arthur A.

1999-01-01

Few identification methods will rapidly or specifically detect all bacteria in the order Chlamydiales, family Chlamydiaceae. In this study, three PCR tests based on sequence data from over 48 chlamydial strains were developed for identification of these bacteria. Two tests exclusively recognized the Chlamydiaceae: a multiplex test targeting the ompA gene and the rRNA intergenic spacer and a TaqMan test targeting the 23S ribosomal DNA. The multiplex test was able to detect as few as 200 inclusion-forming units (IFU), while the TaqMan test could detect 2 IFU. The amplicons produced in these tests ranged from 132 to 320 bp in length. The third test, targeting the 23S rRNA gene, produced a 600-bp amplicon from strains belonging to several families in the order Chlamydiales. Direct sequence analysis of this amplicon has facilitated the identification of new chlamydial strains. These three tests permit ready identification of chlamydiae for diagnostic and epidemiologic study. The specificity of these tests indicates that they might also be used to identify chlamydiae without culture or isolation. PMID:9986815

Evaluation of Alere i RSV for Rapid Detection of Respiratory Syncytial Virus in Children Hospitalized with Acute Respiratory Tract Infection.

PubMed

Peters, Rebecca Marie; Schnee, Sarah Valerie; Tabatabai, Julia; Schnitzler, Paul; Pfeil, Johannes

2017-04-01

Alere i RSV is a novel rapid test which applies a nicking enzyme amplification reaction to detect respiratory syncytial virus in point-of-care settings. In this study, we evaluated the Alere i RSV assay by using frozen nasopharyngeal swab samples that were collected in viral transport medium from children hospitalized with acute respiratory tract infection during the 2015-2016 winter season. Alere i RSV assay results were compared to those for Altona RealStar RSV real-time reverse transcription-PCR (RT-PCR). We found that the overall sensitivity and specificity of the Alere i RSV test was 100% (95% confidence intervals [CI], 93% to 100%) and 97% (95% CI, 89% to 100%), respectively. Positive samples were identified within 5 to 7 min from sample collection. Overall, the Alere i RSV test performed well compared to the RT-PCR assay and has the potential to facilitate the detection of RSV in point-of-care settings. Copyright © 2017 Peters et al.

Acid-fast Smear and Histopathology Results Provide Guidance for the Appropriate Use of Broad-Range Polymerase Chain Reaction and Sequencing for Mycobacteria.

PubMed

Miller, Kennon; Harrington, Susan M; Procop, Gary W

2015-08-01

New molecular diagnostic tests are attractive because of the potential they hold for improving diagnostics in microbiology. The value of these tests, which is often assumed, should be investigated to determine the best use of these potentially powerful tools. To investigate the usefulness of broad-range polymerase chain reaction (PCR), followed by sequencing, in mycobacterial infections. We reviewed the test performance of acid-fast bacilli (AFB) PCR and traditional diagnostic methods (histopathology, AFB smear, and culture). We assessed the diagnostic effect and cost of the unrestricted ordering of broad-range PCR for the detection and identification of mycobacteria in clinical specimens. The AFB PCR was less sensitive than culture and histopathology and was less specific than culture, AFB smear, and histopathology. During 18 months, $93 063 was spent on 183 patient specimens for broad-range PCR and DNA sequencing for mycobacteria to confirm one culture-proven Mycobacterium tuberculosis infection that was also known to be positive by AFB smear and histopathology. In this cohort, there was a false-negative AFB PCR for M tuberculosis and a false-positive AFB PCR for Mycobacterium lentiflavum . Testing of AFB smear-negative specimens from patients without an inflammatory response supportive of a mycobacterial infection is costly and has not been proven to improve patient care. Traditional diagnostics (histopathology, AFB smear, and culture) should remain the primary methods for the detection of mycobacteria in clinical specimens.

Microwave or autoclave treatments destroy the infectivity of infectious bronchitis virus and avian pneumovirus but allow detection by reverse transcriptase-polymerase chain reaction.

PubMed

Elhafi, G; Naylor, C J; Savage, C E; Jones, R C

2004-06-01

A method is described for enabling safe transit of denatured virus samples for polymerase chain reaction (PCR) identification without the risk of unwanted viable viruses. Cotton swabs dipped in avian infectious bronchitis virus (IBV) or avian pneumovirus (APV) were allowed to dry. Newcastle disease virus and avian influenza viruses were used as controls. Autoclaving and microwave treatment for as little as 20 sec destroyed the infectivity of all four viruses. However, both IBV and APV could be detected by reverse transcriptase (RT)-PCR after autoclaving and as long as 5 min microwave treatment (Newcastle disease virus and avian influenza viruses were not tested). Double microwave treatment of IBV and APV with an interval of 2 to 7 days between was tested. After the second treatment, RT-PCR products were readily detected in all samples. Swabs from the tracheas and cloacas of chicks infected with IBV shown to contain infectious virus were microwaved. Swabs from both sources were positive by RT-PCR. Microwave treatment appears to be a satisfactory method of inactivating virus while preserving nucleic acid for PCR identification.

Evaluation of a new single-tube multiprobe real-time PCR for diagnosis of Entamoeba histolytica and Entamoeba dispar.

PubMed

Liang, Shih-Yu; Hsia, Kan-Tai; Chan, Yun-Hsien; Fan, Chia-Kwung; Jiang, Donald Dah-Shyong; Landt, Olfert; Ji, Dar-Der

2010-08-01

A single-tube multiprobe real-time PCR assay for simultaneous detection of Entamoeba histolytica and Entamoeba dispar was developed. One primer pair with 2 species-specific probes was designed based on new SSU RNA regions of the ribosomal DNA-containing episome. The sensitivity is 1 parasite per milliliter of feces and thus superior to the conventional nested PCR and comparable to other published real-time PCR protocols. The applicability for clinical diagnosis was validated with 218 stool specimens from patients. A total of 51 E. histolytica and 39 E. dispar positive samples was detected by the multiprobe real-time PCR compared to 39 and 22 by routine nested PCR diagnosis. The detection rate of Entamoeba species for the multiprobe real-time PCR assays was significantly higher than the nested PCR (40.8% vs. 28.0%, P < 0.01). The test did not show cross reactivity with DNA from Entamoeba moshkovskii, Giardia lamblia , Cryptosporidium sp., Escherichia coli , or other nonpathogenic enteric parasites. The multiprobe real-time PCR assay is simple and rapid and has high specificity and sensitivity. The assay could streamline the laboratory diagnosis procedure and facilitate epidemiological investigation.

Development and validation of a real-time PCR assay for the detection of anguillid herpesvirus 1.

PubMed

van Beurden, S J; Voorbergen-Laarman, M A; Roozenburg, I; van Tellingen, J; Haenen, O L M; Engelsma, M Y

2016-01-01

Anguillid herpesvirus 1 (AngHV1) causes a haemorrhagic disease with increased mortality in wild and farmed European eel, Anguilla anguilla (L.) and Japanese eel Anguilla japonica, Temminck & Schlegel). Detection of AngHV1 is currently based on virus isolation in cell culture, antibody-based typing assays or conventional PCR. We developed, optimized and concisely validated a diagnostic TaqMan probe based real-time PCR assay for the detection of AngHV1. The primers and probe target AngHV1 open reading frame 57, encoding the capsid protease and scaffold protein. Compared to conventional PCR, the developed real-time PCR is faster, less labour-intensive and has a reduced risk of cross-contamination. The real-time PCR assay was shown to be analytically sensitive and specific and has a high repeatability, efficiency and r(2) -value. The diagnostic performance of the assay was determined by testing 10% w/v organ suspensions and virus cultures from wild and farmed European eels from the Netherlands by conventional and real-time PCR. The developed real-time PCR assay is a useful tool for the rapid and sensitive detection of AngHV1 in 10% w/v organ suspensions from wild and farmed European eels. © 2015 John Wiley & Sons Ltd.

Prevalence and molecular characterization of methicillin resistance among Coagulase-negative Staphylococci at a tertiary care center.

PubMed

Bhatt, Puneet; Tandel, Kundan; Singh, Alina; Kumar, M; Grover, Naveen; Sahni, A K

2016-12-01

Methicillin-resistant Coagulase-negative Staphylococci (MR-CoNS) have emerged as an important cause of nosocomial infections especially in patients with prosthetic devices and implants. This study was conducted with an aim to determine the prevalence of methicillin resistance among CoNS isolates at a tertiary care center by both phenotypic and genotypic methods. This cross sectional study was carried out from September 2011 to February 2014 in which 150 non-repetitive clinical isolates of CoNS were identified at the species level by conventional phenotypic methods. Cefoxitin disk (30 μg) diffusion testing was used to determine methicillin resistance and confirmed by detection of mec A gene by polymerase chain reaction (PCR). Out of 150 CoNS isolates, 51 were methicillin resistant by cefoxitin disk diffusion method. Out of these 51 isolates, mec A gene was detected only in 45 isolates. Moreover, mec A gene was also detected in 4 isolates, which were cefoxitin sensitive. Thus, the prevalence of methicillin resistance among CoNS was found to be 32.7% by PCR. The prevalence of methicillin resistance among Coagulase-negative Staphylococci (CoNS) was 32.7% by PCR detection of mec A gene. The sensitivity and specificity of cefoxitin disk diffusion method against mec A gene detection by PCR were found to be more than 90%. It can be concluded from this study that cefoxitin disk diffusion test can be used as a useful screening method to detect methicillin resistance among CoNS isolates. However, detection of mec A gene by PCR remains a more accurate method of detecting methicillin resistance among CoNS.

Species-Specific Detection and Identification of Fusarium Species Complex, the Causal Agent of Sugarcane Pokkah Boeng in China

PubMed Central

Que, Youxiong; Wang, Jihua; Comstock, Jack C.; Wei, Jinjin; McCord, Per H.; Chen, Baoshan; Chen, Rukai; Zhang, Muqing

2014-01-01

Background Pokkah boeng disease caused by the Fusarium species complex results in significant yield losses in sugarcane. Thus, the rapid and accurate detection and identification of the pathogen is urgently required to manage and prevent the spreading of sugarcane pokkah boeng. Methods A total of 101 isolates were recovered from the pokkah boeng samples collected from five major sugarcane production areas in China throughout 2012 and 2013. The causal pathogen was identified by morphological observation, pathogenicity test, and phylogenetic analysis based on the fungus-conserved rDNA-ITS. Species-specific TaqMan real-time PCR and conventional PCR methods were developed for rapid and accurate detection of the causal agent of sugarcane pokkah boeng. The specificity and sensitivity of PCR assay were also evaluated on a total of 84 isolates of Fusarium from China and several isolates from other fungal pathogens of Sporisorium scitamineum and Phoma sp. and sugarcane endophyte of Acremonium sp. Result Two Fusarium species (F. verticillioides and F. proliferatum) that caused sugarcane pokahh boeng were identified by morphological observation, pathogenicity test, and phylogenetic analysis. Species-specific TaqMan PCR and conventional PCR were designed and optimized to target their rDNA-ITS regions. The sensitivity of the TaqMan PCR was approximately 10 pg of fungal DNA input, which was 1,000-fold over conventional PCR, and successfully detected pokkah boeng in the field-grown sugarcane. Conclusions/Significance This study was the first to identify two species, F. verticillioides and F. proliferatum, that were causal pathogens of sugarcane pokkah boeng in China. It also described the development of a species-specific PCR assay to detect and confirm these pathogens in sugarcane plants from mainland China. This method will be very useful for a broad range of research endeavors as well as the regulatory response and management of sugarcane pokkah boeng. PMID:25141192

Comparative Evaluation of Multiplex PCR and Routine Laboratory Phenotypic Methods for Detection of Carbapenemases among Gram Negative Bacilli.

PubMed

Solanki, Rachana; Vanjari, Lavanya; Subramanian, Sreevidya; B, Aparna; E, Nagapriyanka; Lakshmi, Vemu

2014-12-01

Carbapenem resistant pathogens cause infections associated with significant morbidity and mortality. This study evaluates the use of Multiplex PCR for rapid detection of carbapenemase genes among carbapenem resistant Gram negative bacteria in comparison with the existing phenotypic methods like modified Hodge test (MHT), combined disc test (CDT) and automated methods. A total of 100 Carbapenem resistant clinical isolates, [Escherichia coli (25), Klebsiella pneumoniae (35) P. aeruginosa (18) and Acinetobacter baumannii (22)] were screened for the presence of carbapenemases (bla NDM-1, bla VIM , blaIMP and blaKPC genes) by phenotype methods such as the modified Hodge test (MHT) and combined disc test (CDT) and the molecular methods such as Multiplex PCR. Seventy of the 100 isolates were MHT positive while, 65 isolates were positive by CDT. All the CDT positive isolates with EDTA and APB were Metallo betalactamase (MBL) and K. pneumoniae carbapenemase (KPC) producers respectively. bla NDM-1 was present as a lone gene in 44 isolates. In 14 isolates bla NDM-1 gene was present with blaKPC gene, and in one isolate bla NDM-1 gene was present with blaVIM , gene. Only one E. coli isolate had a lone blaKPC gene. We didn't find bla IMP gene in any of the isolates. Neither of the genes could be detected in 35 isolates. Accurate detection of the genes related with carbapenemase production by Molecular methods like Multiplex PCR overcome the limitations of the phenotypic methods and Automated systems.

Influenza and other respiratory virus infections in outpatients with medically attended acute respiratory infection during the 2011-12 influenza season.

PubMed

Zimmerman, Richard K; Rinaldo, Charles R; Nowalk, Mary Patricia; Gk, Balasubramani; Thompson, Mark G; Moehling, Krissy K; Bullotta, Arlene; Wisniewski, Stephen

2014-07-01

Respiratory tract infections are a major cause of outpatient visits, yet only a portion is tested to determine the etiologic organism. Multiplex reverse transcriptase polymerase chain reaction (MRT-PCR) assays for detection of multiple viruses are being used increasingly in clinical settings. During January-April 2012, outpatients with acute respiratory illness (≤ 7 days) were tested for influenza using singleplex RT-PCR (SRT-PCR). A subset was assayed for 18 viruses using MRT-PCR to compare detection of influenza and examine the distribution of viruses and characteristics of patients using multinomial logistic regression. Among 662 participants (6 months-82 years), detection of influenza was similar between the MRT-PCR and SRT-PCR (κ = 0.83). No virus was identified in 267 (40.3%) samples. Commonly detected viruses were human rhinovirus (HRV, 15.4%), coronavirus (CoV, 10.4%), respiratory syncytial virus (RSV, 8.4%), human metapneumovirus (hMPV, 8.3%), and influenza (6%). Co-detections were infrequent (6.9%) and most commonly occurred among those <18 years old. In regression analyses, compared with non-viral illnesses, RSV and hMPV were significantly more frequent in children and less frequent in 18- to 49-year-olds than in those ≥ 50 years (P = 0.01), fever was more common in hMPV and influenza infections (P = 0.008), nasal congestion was more frequent in CoV, HRV, hMPV, influenza and RSV infections (P = 0.001), and body mass index was higher among those with influenza (P = 0.036). Using MRT-PCR, a viral etiology was found in three-fifths of patients with medically attended outpatient visits for acute respiratory illness during the influenza season; co-detected viruses were infrequent. Symptoms varied by viral etiology. © 2014 The Authors. Influenza and Other Respiratory Viruses Published by John Wiley & Sons Ltd.

Highly specific and efficient primers for in-house multiplex PCR detection of Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma hominis and Ureaplasma urealyticum

PubMed Central

2014-01-01

Background Although sophisticated methodologies are available, the use of endpoint polymerase chain reaction (PCR) to detect 16S rDNA genes remains a good approach for estimating the incidence and prevalence of specific infections and for monitoring infections. Considering the importance of the early diagnosis of sexually transmitted infections (STIs), the development of a sensitive and affordable method for identifying pathogens in clinical samples is needed. Highly specific and efficient primers for a multiplex polymerase chain reaction (m-PCR) system were designed in silico to detect the 16S rDNA genes of four bacteria that cause genital infections, and the PCR method was developed. Methods The Genosensor Probe Designer (GPD) (version 1.0a) software was initially used to design highly specific and efficient primers for in-house m-PCR. Single-locus PCR reactions were performed and standardised, and then primers for each locus in turn were added individually in subsequent amplifications until m-PCR was achieved. Amplicons of the expected size were obtained from each of the four bacterial gene fragments. Finally, the analytical specificity and limits of detection were tested. Results Because they did not amplify any product from non-STI tested species, the primers were specific. The detection limits for the Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma hominis and Ureaplasma urealyticum primer sets were 5.12 × 105, 3.9 × 103, 61.19 × 106 and 6.37 × 105 copies of a DNA template, respectively. Conclusions The methodology designed and standardised here could be applied satisfactorily for the simultaneous or individual detection of Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma hominis and Ureaplasma urealyticum. This method is at least as efficient as other previously described methods; however, this method is more affordable for low-income countries. PMID:24997675

Sensitivity and specificity of the AdenoPlus point-of-care system in detecting adenovirus in conjunctivitis patients at an ophthalmic emergency department: a diagnostic accuracy study.

PubMed

Kam, K Y Ronald; Ong, Hon Shing; Bunce, Catey; Ogunbowale, Lola; Verma, Seema

2015-09-01

To estimate the diagnostic accuracy (sensitivity and specificity) of the AdenoPlus point-of-care adenoviral test compared to PCR in an ophthalmic accident and emergency service. These findings were compared with those of a previous study. This was a prospective diagnostic accuracy study on 121 patients presenting to an emergency eye unit with a clinical picture of acute adenoviral conjunctivitis. AdenoPlus testing was carried out on one eye of each patient and a PCR analysis was also performed on a swab taken from the same eye. AdenoPlus and PCR results were interpreted by masked personnel. Sensitivity and specificity for the AdenoPlus test were calculated using PCR results as the reference standard. 121 patients were enrolled and 109 met the inclusion criteria. 43 patients (39.4%) tested positive for adenovirus by PCR analysis. The sensitivity of the AdenoPlus swab in detecting adenovirus was 39.5% (17/43, 95% CI 26% to 54%) and specificity was 95.5% (63/66, 95% CI 87% to 98%) compared to PCR. The AdenoPlus test has a high specificity for diagnosing adenoviral conjunctivitis, but in this clinical setting, we could not reproduce the high sensitivity that has been previously published. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

Highly Sensitive GMO Detection Using Real-Time PCR with a Large Amount of DNA Template: Single-Laboratory Validation.

PubMed

Mano, Junichi; Hatano, Shuko; Nagatomi, Yasuaki; Futo, Satoshi; Takabatake, Reona; Kitta, Kazumi

2018-03-01

Current genetically modified organism (GMO) detection methods allow for sensitive detection. However, a further increase in sensitivity will enable more efficient testing for large grain samples and reliable testing for processed foods. In this study, we investigated real-time PCR-based GMO detection methods using a large amount of DNA template. We selected target sequences that are commonly introduced into many kinds of GM crops, i.e., 35S promoter and nopaline synthase (NOS) terminator. This makes the newly developed method applicable to a wide range of GMOs, including some unauthorized ones. The estimated LOD of the new method was 0.005% of GM maize events; to the best of our knowledge, this method is the most sensitive among the GM maize detection methods for which the LOD was evaluated in terms of GMO content. A 10-fold increase in the DNA amount as compared with the amount used under common testing conditions gave an approximately 10-fold reduction in the LOD without PCR inhibition. Our method is applicable to various analytical samples, including processed foods. The use of other primers and fluorescence probes would permit highly sensitive detection of various recombinant DNA sequences besides the 35S promoter and NOS terminator.

Specific detection of Xanthomonas axonopodis pv. dieffenbachiae in anthurium (Anthurium andreanum) tissues by nested PCR.

PubMed

Robène-Soustrade, Isabelle; Laurent, Philippe; Gagnevin, Lionel; Jouen, Emmanuel; Pruvost, Olivier

2006-02-01

Efficient control of Xanthomonas axonopodis pv. dieffenbachiae, the causal agent of anthurium bacterial blight, requires a sensitive and reliable diagnostic tool. A nested PCR test was developed from a sequence-characterized amplified region marker identified by randomly amplified polymorphic DNA PCR for the detection of X. axonopodis pv. dieffenbachiae. Serological and pathogenicity tests were performed concurrently with the nested PCR test with a large collection of X. axonopodis pv. dieffenbachiae strains that were isolated worldwide and are pathogenic to anthurium and/or other aroids. The internal primer pair directed amplification of the expected product (785 bp) for all 70 X. axonopodis pv. dieffenbachiae strains pathogenic to anthurium tested and for isolates originating from syngonium and not pathogenic to anthurium. This finding is consistent with previous studies which indicated that there is a high level of relatedness between strains from anthurium and strains from syngonium. Strains originating from the two host genera can be distinguished by restriction analysis of the amplification product. No amplification product was obtained with 98 strains of unrelated phytopathogenic bacteria or saprophytic bacteria from the anthurium phyllosphere, except for a weak signal obtained for one X. axonopodis pv. allii strain. Nevertheless, restriction enzyme analysis permitted the two pathovars to be distinguished. The detection threshold obtained with pure cultures or plant extracts (10(3) CFU ml(-1)) allowed detection of the pathogen from symptomless contaminated plants. This test could be a useful diagnostic tool for screening propagation stock plant material and for monitoring international movement of X. axonopodis pv. dieffenbachiae.

Rectal swab sampling followed by an enrichment culture-based real-time PCR assay to detect Salmonella enterocolitis in children.

PubMed

Lin, L-H; Tsai, C-Y; Hung, M-H; Fang, Y-T; Ling, Q-D

2011-09-01

Although routine bacterial culture is the traditional reference standard method for the detection of Salmonella infection in children with diarrhoea, it is a time-consuming procedure that usually only gives results after 3-4 days. Some molecular detection methods can improve the turn-around time to within 24 h, but these methods are not applied directly from stool or rectal swab specimens as routine diagnostic methods for the detection of gastrointestinal pathogens. In this study, we tested the feasibility of a bacterial enrichment culture-based real-time PCR assay method for detecting and screening for diarrhoea in children caused by Salmonella. Our results showed that the minimum real-time PCR assay time required to detect enriched bacterial culture from a swab was 3 h. In all children with suspected Salmonella diarrhoea, the enrichment culture-based real-time PCR achieved 85.4% sensitivity and 98.1% specificity, as compared with the 53.7% sensitivity and 100% specificity of detection with the routine bacterial culture method. We suggest that rectal swab sampling followed by enrichment culture-based real-time PCR is suitable as a rapid method for detecting and screening for Salmonella in paediatric patients. © 2011 The Authors. Clinical Microbiology and Infection © 2011 European Society of Clinical Microbiology and Infectious Diseases.

Prospective Biomarker Screening for Diagnosis of Invasive Aspergillosis in High-Risk Pediatric Patients

PubMed Central

Hafner, Julia; Mengoli, Carlo; Wirth, Clemens; Heussel, Claus Peter; Löffler, Claudia; White, P. Lewis; Ullmann, Andrew J.; Michel, Denise; Wiegering, Verena; Wölfl, Matthias; Schlegel, Paul Gerhardt; Einsele, Hermann; Springer, Jan; Eyrich, Matthias

2016-01-01

ABSTRACT Combined biomarker screening is increasingly used to diagnose invasive aspergillosis (IA) in high-risk patients. In adults, the combination of galactomannan (GM) and fungal DNA detection has proven to be beneficial in the diagnosis of IA. Data in purely pediatric cohorts are scarce. Here, we monitored 39 children shortly before and after allogeneic stem cell transplantation twice weekly by use of a commercial GM enzyme-linked immunosorbent assay (ELISA) and a PCR assay based on amplification of the pan-Aspergillus ITS1/5.8S ribosomal operon. In addition, clinical data were recorded and classification of IA was performed according to the European Organization for the Research and Treatment of Cancer/Mycoses Study Group (EORTC/MSG) criteria. Among the 39 high-risk children, we identified 4 patients (10.3%) with probable and 2 (5.1%) with possible IA. All patients with probable IA were repeatedly positive for both tests (means of 9.5 and 6.8 positive GM and PCR samples, respectively), whereas both possible IA cases were detected by PCR. The sensitivity and specificity were, respectively, 67% and 89% for GM and 100% and 63% for PCR. Positive and negative predictive values were, respectively, 50% and 100% for GM and 27% and 100% for PCR. For the combined testing approach, both values were 100%. The number of positive samples seemed to be lower in patients undergoing antifungal therapy. Sporadically positive tests occurred in 12% (GM) and 42% (PCR) of unclassified patients. In summary, our data show that combined monitoring for GM and fungal DNA also results in a high diagnostic accuracy in pediatric patients. Future studies have to determine whether combined testing is suitable for early detection of subclinical disease and how antifungal prophylaxis impacts assay performance. PMID:27795339

Prospective Biomarker Screening for Diagnosis of Invasive Aspergillosis in High-Risk Pediatric Patients.

PubMed

Loeffler, Juergen; Hafner, Julia; Mengoli, Carlo; Wirth, Clemens; Heussel, Claus Peter; Löffler, Claudia; White, P Lewis; Ullmann, Andrew J; Michel, Denise; Wiegering, Verena; Wölfl, Matthias; Schlegel, Paul Gerhardt; Einsele, Hermann; Springer, Jan; Eyrich, Matthias

2017-01-01

Combined biomarker screening is increasingly used to diagnose invasive aspergillosis (IA) in high-risk patients. In adults, the combination of galactomannan (GM) and fungal DNA detection has proven to be beneficial in the diagnosis of IA. Data in purely pediatric cohorts are scarce. Here, we monitored 39 children shortly before and after allogeneic stem cell transplantation twice weekly by use of a commercial GM enzyme-linked immunosorbent assay (ELISA) and a PCR assay based on amplification of the pan-Aspergillus ITS1/5.8S ribosomal operon. In addition, clinical data were recorded and classification of IA was performed according to the European Organization for the Research and Treatment of Cancer/Mycoses Study Group (EORTC/MSG) criteria. Among the 39 high-risk children, we identified 4 patients (10.3%) with probable and 2 (5.1%) with possible IA. All patients with probable IA were repeatedly positive for both tests (means of 9.5 and 6.8 positive GM and PCR samples, respectively), whereas both possible IA cases were detected by PCR. The sensitivity and specificity were, respectively, 67% and 89% for GM and 100% and 63% for PCR. Positive and negative predictive values were, respectively, 50% and 100% for GM and 27% and 100% for PCR. For the combined testing approach, both values were 100%. The number of positive samples seemed to be lower in patients undergoing antifungal therapy. Sporadically positive tests occurred in 12% (GM) and 42% (PCR) of unclassified patients. In summary, our data show that combined monitoring for GM and fungal DNA also results in a high diagnostic accuracy in pediatric patients. Future studies have to determine whether combined testing is suitable for early detection of subclinical disease and how antifungal prophylaxis impacts assay performance. Copyright © 2016 American Society for Microbiology.

Quantitative PCR: an appropriate tool to detect viable but not culturable Brettanomyces bruxellensis in wine.

PubMed

Willenburg, Elize; Divol, Benoit

2012-11-15

Quantitative PCR as a tool has been used to detect Brettanomyces bruxellensis directly from wine samples. Accurate and timely detection of this yeast is important to prevent unwanted spoilage of wines and beverages. The aim of this study was to distinguish differences between DNA and mRNA as template for the detection of this yeast. The study was also used to determine if it is possible to accurately detect cells in the viable but not culturable (VBNC) state of B. bruxellensis by qPCR. Several methods including traditional plating, epifluorescence counts and qPCR were used to amplify DNA and mRNA. It was observed that mRNA was a better template for the detection in terms of standard curve analysis and qPCR efficiencies. Various primers previously published were tested for their specificity, qPCR efficiency and accuracy of enumeration. A single primer set was selected which amplified a region of the actin-encoding gene. The detection limit for this assay was 10cellsmL(-1). B. bruxellensis could also be quantified in naturally contaminated wines with this assay. The mRNA gave a better indication of the viability of the cells which compared favourably to fluorescent microscopy and traditional cell counts. The ability of the assay to accurately estimate the number of cells in the VBNC state was also demonstrated. Copyright © 2012 Elsevier B.V. All rights reserved.

Development of a TaqMan based real-time PCR assay for detection of Clonorchis sinensis DNA in human stool samples and fishes.

PubMed

Cai, Xian-Quan; Yu, Hai-Qiong; Bai, Jian-Shan; Tang, Jian-Dong; Hu, Xu-Chu; Chen, Ding-Hu; Zhang, Ren-Li; Chen, Mu-Xin; Ai, Lin; Zhu, Xing-Quan

2012-03-01

Clonorchiasis caused by the oriental liver fluke Clonorchis sinensis is a fish-borne zoonosis endemic in a number of countries. This article describes the development of a TaqMan based real-time PCR assay for detection of C. sinensis DNA in human feces and in fishes. Primers targeting the first internal transcribed spacer (ITS-1) sequence of the fluke were highly specific for C. sinensis, as evidenced by the negative amplification of closely related trematodes in the test with the exception of Opisthorchis viverrini. The detection limit of the assay was 1pg of purified genomic DNA, 5EPG (eggs per gram feces) or one metacercaria per gram fish filet. The assay was evaluated by testing 22 human fecal samples and 37 fish tissues microscopically determined beforehand, and the PCR results were highly in agreement with the microscopic results. This real-time PCR assay provides a useful tool for the sensitive detection of C. sinensis DNA in human stool and aquatic samples in China and other endemic countries where O. viverrini and Opisthorchis felineus are absent. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Detection of Herpesvirus anguillae (AngHV-1) in European eel Anguilla anguilla (L.) originating from northern Poland-assessment of suitability of selected diagnostic methods.

PubMed

Nguyen, Tuan Thuc; Jin, Yeonhwa; Kiełpińska, Jolanta; Bergmann, Sven M; Lenk, Matthias; Panicz, Remigiusz

2017-11-01

The Community Action Plan requests EU member states to implement measures that ensure the recovery of the severely depleted European eel stocks. One of the main threats is posed by Anguillid herpesvirus 1 (AngHV-1) leading to increased mortality in both wild and farmed eels. Following recommendations of the OIE to minimize the risk of obtaining false-negative results, the main aim of the study was to optimize diagnostic methods for AngHV-1 detection using conventional PCR, nested PCR and in situ hybridization assay. While 53.3% of the individual organ samples were tested positive for AngHV-1 by PCR, the additional virus analysis via nested PCR revealed that the actual prevalence was 93.3%. In the cell cultivation passages, a cytopathic effect was hardly found in the first two rounds. In the third passage onto cell cultures, a lytic CPE was detected. The identification and confirmation of the viruses obtained from cell cultures as well as directly from the organ tissues were proceeded by PCR, nested PCR and sequencing of the PCR products. While no positive signal was detectable in the first round by PCR using samples from the third cell culture passages, the nested PCR provided weak but visible positive signals. © 2017 John Wiley & Sons Ltd.

Simultaneous detection of three lily viruses using Triplex IC-RT-PCR.

PubMed

Zhang, Yubao; Wang, Yajun; Xie, Zhongkui; Yang, Guo; Guo, Zhihong; Wang, Le

2017-11-01

Viruses commonly infecting lily (Lilium spp.) include: Lily symptomless virus (LSV), Cucumber mosaic virus (CMV) and Lily mottle virus (LMoV). These viruses usually co-infect lilies causing severe economic losses in terms of quantity and quality of flower and bulb production around the world. Reliable and precise detection systems need to be developed for virus identification. We describe the development of a triplex immunocapture (IC) reverse transcription (RT) polymerase chain reaction (PCR) assay for the simultaneous detection of LSV, CMV and LMoV. The triplex IC-RT-PCR was compared with a quadruplex RT-PCR assay. Relative to the quadruplex RT-PCR, the specificity of the triplex IC-RT-PCR system for LSV, CMV and LMoV was 100% for field samples. The sensitivity of the triplex IC-RT-PCR system was 99.4%, 81.4% and 98.7% for LSV, CMV and LMoV, respectively. Agreement (κ) between the results obtained from the two tests was 0.968, 0.844 and 0.984 for LSV, CMV and LMoV, respectively. This is the first report of the simultaneous detection of LSV, CMV and LMoV in a triplex IC-RT-PCR assay. In particular we believe this convenient and reliable triplex IC-RT-PCR method could be used routinely for large-scale field surveys or crop health monitoring of lily. Copyright © 2017. Published by Elsevier B.V.

A pan-Theileria FRET-qPCR survey for Theileria spp. in ruminants from nine provinces of China.

PubMed

Yang, Yi; Mao, Yongjiang; Kelly, Patrick; Yang, Zhangpin; Luan, Lu; Zhang, Jilei; Li, Jing; El-Mahallawy, Heba S; Wang, Chengming

2014-08-31

Theileria spp. are tick transmitted protozoa that can infect large and small ruminants causing disease and economic losses. Diagnosis of infections is often challenging, as parasites can be difficult to detect and identify microscopically and serology is unreliable. While there are PCR assays which can identify certain Theileria spp., there is no one PCR that has been designed to identify all recognized species that occur in ruminants and which will greatly simplify the laboratory diagnoses of infections. Primers and probes for a genus-specific pan-Theileria FRET-qPCR were selected by comparing sequences of recognized Theileria spp. in GenBank and the test validated using reference organisms. The assay was also tested on whole blood samples from large and small ruminants from nine provinces in China. The pan-Theileria FRET-qPCR detected all recognized species but none of the closely related protozoa. In whole blood samples from animals in China, Theileria spp. DNA was detected in 53.2% of the sheep tested (59/111), 44.4% of the goats (120/270) and 30.8% of the cattle (380/1,235). Water buffaloes (n = 29) were negative. Sequencing of some of the PCR products showed cattle in China were infected with T. orientalis/T. sergenti/T. buffeli group while T. ovis and T. luwenshuni were found in sheep and T. luwenshuni in goats. The prevalence of Theileria DNA was significantly higher in Bos p. indicus than in Bos p. taurus (77.7% vs. 18.3%) and copy numbers were also significantly higher (10(4.88) vs. 10(3.00) Theileria 18S rRNA gene copies/per ml whole blood). The pan-Theileria FRET-qPCR can detect all recognized Theileria spp. of ruminants in a single reaction. Large and small ruminants in China are commonly infected with a variety of Theileria spp.

Comparative quantification of human intestinal bacteria based on cPCR and LDR/LCR

PubMed Central

Tang, Zhou-Rui; Li, Kai; Zhou, Yu-Xun; Xiao, Zhen-Xian; Xiao, Jun-Hua; Huang, Rui; Gu, Guo-Hao

2012-01-01

AIM: To establish a multiple detection method based on comparative polymerase chain reaction (cPCR) and ligase detection reaction (LDR)/ligase chain reaction (LCR) to quantify the intestinal bacterial components. METHODS: Comparative quantification of 16S rDNAs from different intestinal bacterial components was used to quantify multiple intestinal bacteria. The 16S rDNAs of different bacteria were amplified simultaneously by cPCR. The LDR/LCR was examined to actualize the genotyping and quantification. Two beneficial (Bifidobacterium, Lactobacillus) and three conditionally pathogenic bacteria (Enterococcus, Enterobacterium and Eubacterium) were used in this detection. With cloned standard bacterial 16S rDNAs, standard curves were prepared to validate the quantitative relations between the ratio of original concentrations of two templates and the ratio of the fluorescence signals of their final ligation products. The internal controls were added to monitor the whole detection flow. The quantity ratio between two bacteria was tested. RESULTS: cPCR and LDR revealed obvious linear correlations with standard DNAs, but cPCR and LCR did not. In the sample test, the distributions of the quantity ratio between each two bacterial species were obtained. There were significant differences among these distributions in the total samples. But these distributions of quantity ratio of each two bacteria remained stable among groups divided by age or sex. CONCLUSION: The detection method in this study can be used to conduct multiple intestinal bacteria genotyping and quantification, and to monitor the human intestinal health status as well. PMID:22294830

Comparative quantification of human intestinal bacteria based on cPCR and LDR/LCR.

PubMed

Tang, Zhou-Rui; Li, Kai; Zhou, Yu-Xun; Xiao, Zhen-Xian; Xiao, Jun-Hua; Huang, Rui; Gu, Guo-Hao

2012-01-21

To establish a multiple detection method based on comparative polymerase chain reaction (cPCR) and ligase detection reaction (LDR)/ligase chain reaction (LCR) to quantify the intestinal bacterial components. Comparative quantification of 16S rDNAs from different intestinal bacterial components was used to quantify multiple intestinal bacteria. The 16S rDNAs of different bacteria were amplified simultaneously by cPCR. The LDR/LCR was examined to actualize the genotyping and quantification. Two beneficial (Bifidobacterium, Lactobacillus) and three conditionally pathogenic bacteria (Enterococcus, Enterobacterium and Eubacterium) were used in this detection. With cloned standard bacterial 16S rDNAs, standard curves were prepared to validate the quantitative relations between the ratio of original concentrations of two templates and the ratio of the fluorescence signals of their final ligation products. The internal controls were added to monitor the whole detection flow. The quantity ratio between two bacteria was tested. cPCR and LDR revealed obvious linear correlations with standard DNAs, but cPCR and LCR did not. In the sample test, the distributions of the quantity ratio between each two bacterial species were obtained. There were significant differences among these distributions in the total samples. But these distributions of quantity ratio of each two bacteria remained stable among groups divided by age or sex. The detection method in this study can be used to conduct multiple intestinal bacteria genotyping and quantification, and to monitor the human intestinal health status as well.

Development of a novel real-time RT-PCR assay to detect Seneca Valley virus-1 associated with emerging cases of vesicular disease in pigs.

PubMed

Fowler, Veronica L; Ransburgh, Russell H; Poulsen, Elizabeth G; Wadsworth, Jemma; King, Donald P; Mioulet, Valerie; Knowles, Nick J; Williamson, Susanna; Liu, Xuming; Anderson, Gary A; Fang, Ying; Bai, Jianfa

2017-01-01

Seneca Valley virus 1 (SVV-1) can cause vesicular disease that is clinically indistinguishable from foot-and-mouth disease, vesicular stomatitis and swine vesicular disease. SVV-1-associated disease has been identified in pigs in several countries, namely USA, Canada, Brazil and China. Diagnostic tests are required to reliably detect this emerging virus, and this report describes the development and evaluation of a novel real-time (r) reverse-transcription (RT) PCR assay (rRT-PCR), targeting the viral polymerase gene (3D) of SVV-1. This new assay detected all historical and contemporary SVV-1 isolates examined (n=8), while no cross-reactivity was observed with nucleic acid templates prepared from other vesicular disease viruses or common swine pathogens. The analytical sensitivity of the rRT-PCR was 0.79 TCID 50 /ml and the limit of detection was equivalent using two different rRT-PCR master-mixes. The performance of the test was further evaluated using pig nasal (n=25) and rectal swab samples (n=25), where concordant results compared to virus sequencing were generated for 43/50 samples. The availability of this assay, will enable laboratories to rapidly detect SVV-1 in cases of vesicular disease in pigs, negated for notifiable diseases, and could enable existing knowledge gaps to be investigated surrounding the natural epidemiology of SVV-1. Copyright © 2016 Elsevier B.V. All rights reserved.

A multiplex real-time PCR assay for identification of Pneumocystis jirovecii, Histoplasma capsulatum, and Cryptococcus neoformans/Cryptococcus gattii in samples from AIDS patients with opportunistic pneumonia.

PubMed

Gago, Sara; Esteban, Cristina; Valero, Clara; Zaragoza, Oscar; Puig de la Bellacasa, Jorge; Buitrago, María José

2014-04-01

A molecular diagnostic technique based on real-time PCR was developed for the simultaneous detection of three of the most frequent causative agents of fungal opportunistic pneumonia in AIDS patients: Pneumocystis jirovecii, Histoplasma capsulatum, and Cryptococcus neoformans/Cryptococcus gattii. This technique was tested in cultured strains and in clinical samples from HIV-positive patients. The methodology used involved species-specific molecular beacon probes targeted to the internal transcribed spacer regions of the rDNA. An internal control was also included in each assay. The multiplex real-time PCR assay was tested in 24 clinical strains and 43 clinical samples from AIDS patients with proven fungal infection. The technique developed showed high reproducibility (r(2) of >0.98) and specificity (100%). For H. capsulatum and Cryptococcus spp., the detection limits of the method were 20 and 2 fg of genomic DNA/20 μl reaction mixture, respectively, while for P. jirovecii the detection limit was 2.92 log10 copies/20 μl reaction mixture. The sensitivity in vitro was 100% for clinical strains and 90.7% for clinical samples. The assay was positive for 92.5% of the patients. For one of the patients with proven histoplasmosis, P. jirovecii was also detected in a bronchoalveolar lavage sample. No PCR inhibition was detected. This multiplex real-time PCR technique is fast, sensitive, and specific and may have clinical applications.

Multiplex real-time PCR assay for Legionella species.

PubMed

Kim, Seung Min; Jeong, Yoojung; Sohn, Jang Wook; Kim, Min Ja

2015-12-01

Legionella pneumophila serogroup 1 (sg1) accounts for the majority of infections in humans, but other Legionella species are also associated with human disease. In this study, a new SYBR Green I-based multiplex real-time PCR assay in a single reaction was developed to allow the rapid detection and differentiation of Legionella species by targeting specific gene sequences. Candidate target genes were selected, and primer sets were designed by referring to comparative genomic hybridization data of Legionella species. The Legionella species-specific groES primer set successfully detected all 30 Legionella strains tested. The xcpX and rfbA primers specifically detected L. pneumophila sg1-15 and L. pneumophila sg1, respectively. In addition, this assay was validated by testing clinical samples and isolates. In conclusion, this novel multiplex real-time PCR assay might be a useful diagnostic tool for the rapid detection and differentiation of Legionella species in both clinical and epidemiological studies. Copyright © 2015 Elsevier Ltd. All rights reserved.

Development of Quantitative Real-Time PCR Assays for Detection and Quantification of Surrogate Biological Warfare Agents in Building Debris and Leachate▿

PubMed Central

Saikaly, Pascal E.; Barlaz, Morton A.; de los Reyes, Francis L.

2007-01-01

Evaluation of the fate and transport of biological warfare (BW) agents in landfills requires the development of specific and sensitive detection assays. The objective of the current study was to develop and validate SYBR green quantitative real-time PCR (Q-PCR) assays for the specific detection and quantification of surrogate BW agents in synthetic building debris (SBD) and leachate. Bacillus atrophaeus (vegetative cells and spores) and Serratia marcescens were used as surrogates for Bacillus anthracis (anthrax) and Yersinia pestis (plague), respectively. The targets for SYBR green Q-PCR assays were the 16S-23S rRNA intergenic transcribed spacer (ITS) region and recA gene for B. atrophaeus and the gyrB, wzm, and recA genes for S. marcescens. All assays showed high specificity when tested against 5 ng of closely related Bacillus and Serratia nontarget DNA from 21 organisms. Several spore lysis methods that include a combination of one or more of freeze-thaw cycles, chemical lysis, hot detergent treatment, bead beat homogenization, and sonication were evaluated. All methods tested showed similar threshold cycle values. The limit of detection of the developed Q-PCR assays was determined using DNA extracted from a pure bacterial culture and DNA extracted from sterile water, leachate, and SBD samples spiked with increasing quantities of surrogates. The limit of detection for B. atrophaeus genomic DNA using the ITS and B. atrophaeus recA Q-PCR assays was 7.5 fg per PCR. The limits of detection of S. marcescens genomic DNA using the gyrB, wzm, and S. marcescens recA Q-PCR assays were 7.5 fg, 75 fg, and 7.5 fg per PCR, respectively. Quantification of B. atrophaeus vegetative cells and spores was linear (R2 > 0.98) over a 7-log-unit dynamic range down to 101 B. atrophaeus cells or spores. Quantification of S. marcescens (R2 > 0.98) was linear over a 6-log-unit dynamic range down to 102 S. marcescens cells. The developed Q-PCR assays are highly specific and sensitive and can be used for monitoring the fate and transport of the BW surrogates B. atrophaeus and S. marcescens in building debris and leachate. PMID:17720820

A Multiplex PCR/LDR Assay for Simultaneous Detection and Identification of the NIAID Category B Bacterial Food and Water-borne Pathogens

PubMed Central

Rundell, Mark S.; Pingle, Maneesh; Das, Sanchita; Hussain, Aashiq; Ocheretina, Oksana; Charles, Macarthur; Larone, Davise H.; Spitzer, Eric D.; Golightly, Linnie; Barany, Francis

2014-01-01

Enteric pathogens that cause gastroenteritis remain a major global health concern. The goal of this study was to develop a multiplex PCR/LDR assay for the detection of all NIAID category B bacterial food and water-borne pathogens directly from stool specimens. To validate the PCR/LDR assay, clinical isolates of Campylobacter spp., Vibrio spp., Shigella spp., Salmonella spp., Listeria monocytogenes, Yersinia enterocolitica, and diarrheagenic Escherichia coli were tested. The sensitivity and specificity of the assay was assessed using a large number of seeded culture-negative stool specimens and a smaller set of clinical specimens from Haiti. The overall sensitivity ranged from 91 to 100% (median 100%) depending on the species. For the majority of organisms the sensitivity was 100%. The overall specificity based on initial testing ranged from 98% to 100% depending on the species. After additional testing of discordant samples the lowest specificity was 99.4%. PCR/LDR detected additional category B agents (particularly diarrheagenic E. coli) in 11/40 specimens from Haiti that were culture-positive for V. cholerae and in approximately 1% of routine culture-negative stool specimens from a hospital in New York. This study demonstrated the ability of the PCR/LDR assay to detect a large comprehensive panel of category B enteric bacterial pathogens as well as mixed infections. This type of assay has the potential to provide earlier warnings of possible public health threats and more accurate surveillance of food and water-borne pathogens. PMID:24709368

Evaluation of Immunomagnetic Separation Method for the Recovery of Hepatitis A Virus and GI.1 and GII.4 Norovirus Strains Seeded on Oyster and Mussel.

PubMed

Ha, Ji-Hyoung; Choi, Changsun; Ha, Sang-Do

2014-12-01

Outbreaks of viral diseases are frequently associated with the consumption of minimally processed shellfish. Among the viruses in these outbreaks, hepatitis A virus (HAV) and human norovirus (NoV) have been increasingly reported as the most common food-borne pathogens. These viruses must be concentrated in tested samples in order to be detected. In this study, a method for the detection of NoV and HAV in shellfish using an immuno-magnetic separation (IMS) procedure combined with reverse transcriptase (RT)-PCR was developed. The IMS/RT-PCR method was applied to investigate the recovery rates of HAV, NoV GI.1, and GII.4 from oyster and mussel. Based on IMS/RT-PCR results, recovery rates for HAV from oyster and mussel test samples were 2.4 and 1.1%, respectively. The NoV GI.1 recovery rates from oyster and mussel samples were 4.9-9.2% (mean 6.9%) and 4.3-8.6% (mean 6.2%), respectively, and the NoV GII.4 recovery rates were 8.8 and 8.5%, respectively. These results verified that HAV, NoV GI.1, and GII.4 can be detected in all the test samples using the IMS/RT-PCR method, although the three inoculated viruses were recovered with low efficiency. In conclusion, the IMS/RT-PCR method can be used to efficiently and rapidly detect viruses such as HAV and NoV in shellfish such as oyster and mussel.

Diagnosis of EML4-ALK Translocation With FISH, Immunohistochemistry, and Real-time Polymerase Chain Reaction in Patients With Non-Small Cell Lung Cancer.

PubMed

Cruz-Rico, Graciela; Avilés-Salas, Alejandro; Segura-González, Manuel; Espinosa-García, Ana María; Ramírez-Tirado, Laura Alejandra; Morales-Oyarvide, Vicente; Rojas-Marín, Carlos; Cardona, Andrés-Felipe; Arrieta, Oscar

2017-12-01

To assess anaplastic lymphoma kinase (ALK) rearrangement detection with immunohistochemistry (IHC) and real-time polymerase chain reaction (RT-qPCR) in comparison with fluorescence in situ hybridization (FISH). Tumor tissue samples from 230 patients with advanced non-small cell lung cancer (NSCLC) were analyzed by FISH to detect ALK rearrangements. Additional IHC tests using 5A4 clone and RT-qPCR (variants 1 to 5) were performed in 63 and 48 patients, respectively. Thirteen percent of FISH tests were not evaluable. From the remaining tests (n=200), 18 (9.0%) were ALK positive (ALK). ALK patients were significantly younger at the time of diagnosis (below 55 y, 14.3% vs. 5.5%, P=0.035), were light smokers (tobacco index <10, 12.6% vs. 4.1%, P=0.049), and presented adenocarcinoma with a mucinous component (30.8 vs. 8.0%, P=0.007). When comparing FISH with IHC using a cutoff of 1+ or 2+, and only 2+ staining intensity, the sensitivity, specificity, negative predictive value, and positive predictive value were as follows: 83.3%, 100.0%, 93.75%, and 100.0%; and 55.6%, 100.0%, 84.9%, and 100.0%, respectively. For RT-qPCR, these results were 55.6, 100, 90.7, and 100.0%, respectively. Our results suggest that RT-qPCR is an inadequate initial test for detecting ALK-positive lung cancer. IHC is highly useful as an initial screening test for ALK rearrangement detection in NSCLC. These results contribute to the medical literature on the establishment of IHC as a standard diagnostic test for ALK rearrangements in NSCLC.

Molecular diagnosis of Salmonella typhi and its virulence in suspected typhoid blood samples through nested multiplex PCR.

PubMed

Prabagaran, Solai Ramatchandirane; Kalaiselvi, Vellingiri; Chandramouleeswaran, Naganathan; Deepthi, Krishnan Nair Geetha; Brahmadathan, Kootallur Narayanan; Mani, Mariappa

2017-08-01

A nested multiplex polymerase chain reaction (PCR) based diagnosis was developed for the detection of virulent Salmonella typhi in the blood specimens from patients suspected for typhoid fever. After the Widal test, two pairs of primers were used for the detection of flagellin gene (fliC) of S. typhi. Among them, those positive for fliC alone were subjected to identification of genes in Via B operon of Salmonella Pathogenesity Island (SPI-7) where four primer pairs were used to detect tviA and tviB genes. Among 250 blood samples tested, 115 were positive by fliC PCR; 22 of these were negative for tviA and tviB. Hence, the method described here can be used to diagnose the incidence of Vi-negative serovar typhi especially in endemic regions where the Vi vaccine is administered. Copyright © 2017 Elsevier B.V. All rights reserved.

Inter-laboratory analysis of selected genetically modified plant reference materials with digital PCR.

PubMed

Dobnik, David; Demšar, Tina; Huber, Ingrid; Gerdes, Lars; Broeders, Sylvia; Roosens, Nancy; Debode, Frederic; Berben, Gilbert; Žel, Jana

2018-01-01

Digital PCR (dPCR), as a new technology in the field of genetically modified (GM) organism (GMO) testing, enables determination of absolute target copy numbers. The purpose of our study was to test the transferability of methods designed for quantitative PCR (qPCR) to dPCR and to carry out an inter-laboratory comparison of the performance of two different dPCR platforms when determining the absolute GM copy numbers and GM copy number ratio in reference materials certified for GM content in mass fraction. Overall results in terms of measured GM% were within acceptable variation limits for both tested dPCR systems. However, the determined absolute copy numbers for individual genes or events showed higher variability between laboratories in one third of the cases, most possibly due to variability in the technical work, droplet size variability, and analysis of the raw data. GMO quantification with dPCR and qPCR was comparable. As methods originally designed for qPCR performed well in dPCR systems, already validated qPCR assays can most generally be used for dPCR technology with the purpose of GMO detection. Graphical abstract The output of three different PCR-based platforms was assessed in an inter-laboratory comparison.

Development of duplex real-time PCR for the detection of WSSV and PstDV1 in cultivated shrimp.

PubMed

Leal, Carlos A G; Carvalho, Alex F; Leite, Rômulo C; Figueiredo, Henrique C P

2014-07-05

The White spot syndrome virus (WSSV) and Penaeus stylirostris penstyldensovirus 1 (previously named Infectious hypodermal and hematopoietic necrosis virus-IHHNV) are two of the most important viral pathogens of penaeid shrimp. Different methods have been applied for diagnosis of these viruses, including Real-time PCR (qPCR) assays. A duplex qPCR method allows the simultaneous detection of two viruses in the same sample, which is more cost-effective than assaying for each virus separately. Currently, an assay for the simultaneous detection of the WSSV and the PstDV1 in shrimp is unavailable. The aim of this study was to develop and standardize a duplex qPCR assay for the simultaneous detection of the WSSV and the PstDV1 in clinical samples of diseased L. vannamei. In addition, to evaluate the performance of two qPCR master mixes with regard to the clinical sensitivity of the qPCR assay, as well as, different methods for qPCR results evaluation. The duplex qPCR assay for detecting WSSV and PstDV1 in clinical samples was successfully standardized. No difference in the amplification of the standard curves was observed between the duplex and singleplex assays. Specificities and sensitivities similar to those of the singleplex assays were obtained using the optimized duplex qPCR. The analytical sensitivities of duplex qPCR were two copies of WSSV control plasmid and 20 copies of PstDV1 control plasmid. The standardized duplex qPCR confirmed the presence of viral DNA in 28 from 43 samples tested. There was no difference for WSSV detection using the two kits and the distinct methods for qPCR results evaluation. High clinical sensitivity for PstDV1 was obtained with TaqMan Universal Master Mix associated with relative threshold evaluation. Three cases of simultaneous infection by the WSSV and the PstDV1 were identified with duplex qPCR. The standardized duplex qPCR was shown to be a robust, highly sensitive, and feasible diagnostic tool for the simultaneous detection of the WSSV and the PstDV1 in whiteleg shrimp. The use of the TaqMan Universal Master Mix and the relative threshold method of data analysis in our duplex qPCR method provided optimal levels of sensitivity and specificity.

Development of SYBR Green and TaqMan quantitative real-time PCR assays for hepatopancreatic parvovirus (HPV) infecting Penaeus monodon in India.

PubMed

Yadav, Reena; Paria, Anutosh; Mankame, Smruti; Makesh, M; Chaudhari, Aparna; Rajendran, K V

2015-12-01

Hepatopancreatic parvovirus (HPV) infects Penaeus monodon and causes mortality in the larval stages. Further, it has been implicated in the growth retardation in cultured P. monodon. Though different geographical isolates of HPV show large sequence variations, a sensitive PCR assay specific to Indian isolate has not yet been reported. Here, we developed a sensitive SYBR Green-based and TaqMan real-time PCR for the detection and quantification of the virus. A 441-bp PCR amplicon was cloned in pTZ57 R/T vector and the plasmid copy number was estimated. A 10-fold serial dilution of the plasmid DNA from 1 × 10(9) copies to 1 copy was prepared and used as the standard. The primers were tested initially using the standard on a conventional PCR format to determine the linearity of detection. The standards were further tested on real-time PCR format using SYBR Green and TaqMan chemistry and standard curves were generated based on the Ct values from three well replicates for each dilution. The assays were found to be sensitive, specific and reproducible with a wide dynamic range (1 × 10(9) to 10 copies) with coefficient of regression (R(2)) > 0.99, calculated average slope -3.196 for SYBR Green assay whereas, for TaqMan assay it was >0.99 and -3.367, respectively. The intra- and inter-assay variance of the Ct values ranged from 0.26% to 0.94% and 0.12% to 0.81%, respectively, for SYBR Green assay, and the inter-assay variance of the Ct values for TaqMan assay ranged from 0.07% to 1.93%. The specificity of the assays was proved by testing other DNA viruses of shrimp such as WSSV, IHHNV and MBV. Standardized assays were further tested to detect and quantify HPV in the post-larvae of P. monodon. The result was further compared with conventional PCR to test the reproducibility of the test. The assay was also used to screen Litopeneaus vannamei, Macrobrachium rosenbergii and Scylla serrata for HPV. Copyright © 2015 Elsevier Ltd. All rights reserved.

Molecular Diagnosis of Chagas Disease in Colombia: Parasitic Loads and Discrete Typing Units in Patients from Acute and Chronic Phases

PubMed Central

Hernández, Carolina; Cucunubá, Zulma; Flórez, Carolina; Olivera, Mario; Valencia, Carlos; Zambrano, Pilar; León, Cielo; Ramírez, Juan David

2016-01-01

Background The diagnosis of Chagas disease is complex due to the dynamics of parasitemia in the clinical phases of the disease. The molecular tests have been considered promissory because they detect the parasite in all clinical phases. Trypanosoma cruzi presents significant genetic variability and is classified into six Discrete Typing Units TcI-TcVI (DTUs) with the emergence of foreseen genotypes within TcI as TcIDom and TcI Sylvatic. The objective of this study was to determine the operating characteristics of molecular tests (conventional and Real Time PCR) for the detection of T. cruzi DNA, parasitic loads and DTUs in a large cohort of Colombian patients from acute and chronic phases. Methodology/Principal Findings Samples were obtained from 708 patients in all clinical phases. Standard diagnosis (direct and serological tests) and molecular tests (conventional PCR and quantitative PCR) targeting the nuclear satellite DNA region. The genotyping was performed by PCR using the intergenic region of the mini-exon gene, the 24Sa, 18S and A10 regions. The operating capabilities showed that performance of qPCR was higher compared to cPCR. Likewise, the performance of qPCR was significantly higher in acute phase compared with chronic phase. The median parasitic loads detected were 4.69 and 1.33 parasite equivalents/mL for acute and chronic phases. The main DTU identified was TcI (74.2%). TcIDom genotype was significantly more frequent in chronic phase compared to acute phase (82.1% vs 16.6%). The median parasitic load for TcIDom was significantly higher compared with TcI Sylvatic in chronic phase (2.58 vs.0.75 parasite equivalents/ml). Conclusions/Significance The molecular tests are a precise tool to complement the standard diagnosis of Chagas disease, specifically in acute phase showing high discriminative power. However, it is necessary to improve the sensitivity of molecular tests in chronic phase. The frequency and parasitemia of TcIDom genotype in chronic patients highlight its possible relationship to the chronicity of the disease. PMID:27648938

Development of a test for bovine tuberculosis in cattle based on measurement of gamma interferon mRNA by real-time PCR.

PubMed

Gan, W; Zhou, X; Yang, H; Chen, H; Qiao, J; Khan, S H; Yang, L; Yin, X; Zhao, D

2013-08-03

The infection status of cattle for bovine tuberculosis (bTB) was determined by real-time PCR, comparing the levels of IFN-γ mRNA in blood cultures stimulated with either bovine or avian tuberculin with non-stimulated control (phosphate buffer saline, PBS) blood culture. Totally, 137 cattle were tested to validate the assay, in which 54 were IFN-γ real-time quantitative PCR (RT-qPCR) positive, while the remaining 83 were found negative. Meanwhile, the IFN-γ ELISA test was carried out using the Bovigam IFN-γ detection ELISA kit and these results were used as a standard. The results of the single intradermal tuberculin tests (SIDT) and IFN-γ RT-qPCR tests were compared and revealed that the RT-qPCR correlated better with the ELISA and its accuracy was higher than SIDT. This indicates the RT-qPCR is a useful diagnostic method for bTB in cattle. However, several limitations remain for our approach, such as lack of a TB lesions or postmortem test results as a gold standard. Further improvements should be made in the future to increase accuracy of diagnosis of bTB in cattle.

Urinary PCR as an increasingly useful tool for an accurate diagnosis of leptospirosis in livestock.

PubMed

Hamond, C; Martins, G; Loureiro, A P; Pestana, C; Lawson-Ferreira, R; Medeiros, M A; Lilenbaum, W

2014-03-01

The aim of the present study was to consider the wide usage of urinary PCR as an increasingly useful tool for an accurate diagnosis of leptospirosis in livestock. A total of 512 adult animals (300 cattle, 138 horses, 59 goats and 15 pigs), from herds/flocks with reproductive problems in Rio de Janeiro, Brazil was studied by serology and urinary PCR. From the 512 serum samples tested, 223 (43.5 %) were seroreactive (cattle: 45.6 %, horses: 41.3 %, goats: 34%and pigs: 60 %). PCR detected leptospiral DNA in 32.4 % (cattle: 21.6 %, horses: 36.2 %, goats: 77.4 % and pigs: 33.3 %. To our knowledge there is no another study including such a large number of samples (512) from different species, providing a comprehensive analysis of the usage of PCR for detecting leptospiral carriers in livestock. Serological and molecular results were discrepant, regardless the titre, what was an expected outcome. Nevertheless, it is impossible to establish agreement between these tests, since the two methodologies are conducted on different samples (MAT - serum; PCR - urine). Additionally, the MAT is an indirect method and PCR is a direct one. In conclusion, we have demonstrated that urinary PCR should be considered and encouraged as an increasingly useful tool for an accurate diagnosis of leptospirosis in livestock.

A simplified strategy for sensitive detection of Rose rosette virus compatible with three RT-PCR chemistries.

PubMed

Dobhal, Shefali; Olson, Jennifer D; Arif, Mohammad; Garcia Suarez, Johnny A; Ochoa-Corona, Francisco M

2016-06-01

Rose rosette disease is a disorder associated with infection by Rose rosette virus (RRV), a pathogen of roses that causes devastating effects on most garden cultivated varieties, and the wild invasive rose especially Rosa multiflora. Reliable and sensitive detection of this disease in early phases is needed to implement proper control measures. This study assesses a single primer-set based detection method for RRV and demonstrates its application in three different chemistries: Endpoint RT-PCR, TaqMan-quantitative RT-PCR (RT-qPCR) and SYBR Green RT-qPCR with High Resolution Melting analyses. A primer set (RRV2F/2R) was designed from consensus sequences of the nucleocapsid protein gene p3 located in the RNA 3 region of RRV. The specificity of primer set RRV2F/2R was validated in silico against published GenBank sequences and in-vitro against infected plant samples and an exclusivity panel of near-neighbor and other viruses that commonly infect Rosa spp. The developed assay is sensitive with a detection limit of 1fg from infected plant tissue. Thirty rose samples from 8 different states of the United States were tested using the developed methods. The developed methods are sensitive and reliable, and can be used by diagnostic laboratories for routine testing and disease management decisions. Copyright © 2016 Elsevier B.V. All rights reserved.

Comparison of three DNA extraction methods for the detection and quantification of GMO in Ecuadorian manufactured food.

PubMed

Pacheco Coello, Ricardo; Pestana Justo, Jorge; Factos Mendoza, Andrés; Santos Ordoñez, Efrén

2017-12-20

In Ecuador, food products need to be labeled if exceeded 0.9% of transgenic content in whole products. For the detection of genetically modified organisms (GMOs), three DNA extraction methods were tested in 35 food products commercialized in Ecuador. Samples with positive amplification of endogenous genes were screened for the presence of the Cauliflower mosaic virus 35S-promoter (P35S) and the nopaline synthase-terminator (Tnos). TaqMan™ probes were used for determination of transgenic content of the GTS 40-3-2 and MON810 events through quantitative PCR (qPCR). Twenty-six processed food samples were positive for the P35S alone and eight samples for the Tnos and P35S. Absolute qPCR results indicated that eleven samples were positive for GTS 40-3-2 specific event and two for MON810 specific event. A total of nine samples for events GTS 40-3-2 and MON810 exceeded the umbral allowed of transgenic content in the whole food product with the specific events. Different food products may require different DNA extraction protocols for GMO detection through PCR. Among the three methods tested, the DNeasy mericon food kit DNA extraction method obtained higher proportion of amplified endogenous genes through PCR. Finally, event-specific GMOs were detected in food products in Ecuador.

Synovial fluid multiplex PCR is superior to culture for detection of low-virulent pathogens causing periprosthetic joint infection.

PubMed

Morgenstern, Christian; Cabric, Sabrina; Perka, Carsten; Trampuz, Andrej; Renz, Nora

2018-02-01

Analysis of joint aspirate is the standard preoperative investigation for diagnosis of periprosthetic joint infection (PJI). We compared the diagnostic performance of culture and multiplex polymerase chain reaction (PCR) of synovial fluid for diagnosis of PJI. Patients in whom aspiration of the prosthetic hip or knee joint was performed before revision arthroplasty were prospectively included. The performance of synovial fluid culture and multiplex PCR was compared by McNemar's chi-squared test. A total of 142 patients were included, 82 with knee and 60 with hip prosthesis. PJI was diagnosed in 77 patients (54%) and aseptic failure in 65 patients (46%). The sensitivity of synovial fluid culture and PCR was 52% and 60%, respectively, showing concordant results in 116 patients (82%). In patients with PJI, PCR missed 6 high-virulent pathogens (S. aureus, streptococci, E. faecalis, E. coli) which grew in synovial fluid culture, whereas synovial fluid culture missed 12 pathogens detected by multiplex PCR, predominantly low-virulent pathogens (Cutibacterium acnes and coagulase-negative staphylococci). In patients with aseptic failure, PCR detected 6 low-virulent organisms (predominantly C. acnes). While the overall performance of synovial fluid PCR was comparable to culture, PCR was superior for detection of low-virulent bacteria such as Cutibacterium spp. and coagulase-negative staphylococci. In addition, synovial fluid culture required several days for growth, whereas multiplex PCR provided results within 5hours in an automated manner. Copyright © 2017 Elsevier Inc. All rights reserved.

Real-time PCR detection of Brucella spp. DNA in lesions and viscera of bovine carcasses.

PubMed

Sola, Marília Cristina; da Veiga Jardim, Eurione A G; de Freitas, Marcius Ribeiro; de Mesquita, Albenones José

2014-09-01

This study reports a real-time PCR assay for the detection of Brucella spp. associated with the FTA® Elute method in lesions observed during sanitary inspections in beef slaughter. Of the total 276 samples, 78 (28.3%) tested positive and 198 (71.7%) negative for Brucella spp. The real-time PCR technique associated with the FTA® Elute method proved to be an important tool for the diagnosis, judgment about and disposal of carcasses and viscera of slaughtered animals. Copyright © 2014 Elsevier B.V. All rights reserved.

[Long distance-PCR for detection of factor VIII gene inversion in patients with severe hemophilia A].

PubMed

Ding, Pei-Fang; Sun, Wei-Sheng; Wang, Qin-You; Liu, De-Chun; Zhang, Xue-Qin; Teng, Bin; Shen, Fa-Kui

2003-08-01

The aim of current study was to detect intron 22 inversion of factor VIII gene in severe hemophilia A (HA) patients and screen the carriers of the gene inversion. Fifty-five cases of severe HA were involved and factor VIII gene inversion was detected and identified by long distance-PCR (LD-PCR) and 0.6% agarose gel electrophoresis. The 11 kb and 12 kb bands indicate the factor VIII gene inversion and non-inversion, respectively. Occurring of both 11 kb and 12 kb bands indicates a carrier of the inversion. The results showed that factor VIII gene inversion existed in 22 out of 55 cases, which accounted for about 40% of total detected patients. Five carriers of factor VIII gene inversion were diagnosed from the members in 15 families. In conclusion, LD-PCR assay is a simple, rapid and accurate method for detection of factor VIII gene inversion, and this approach is helpful in screening, carrier testing, and prenatal diagnosis of severe hemophilia A.

Multiplex DNA detection of food allergens on a digital versatile disk.

PubMed

Tortajada-Genaro, Luis A; Santiago-Felipe, Sara; Morais, Sergi; Gabaldón, José Antonio; Puchades, Rosa; Maquieira, Ángel

2012-01-11

The development of a DNA microarray method on a digital versatile disk (DVD) is described for the simultaneous detection of traces of hazelnut ( Corylus avellana L.), peanut ( Arachis hypogaea ), and soybean ( Glycine max ) in foods. After DNA extraction, multiplex PCR was set up using 5'-labeled specific primers for Cor a 1, Ar h 2, and Le genes, respectively. Digoxin-labeled PCR products were detected by hybridization with 5'-biotinylated probes immobilized on a streptavidin-modified DVD surface. The reaction product attenuates the signal intensity of the laser that reached the DVD drive used as detector, correlating well with the amount of amplified sequence. Analytical performances showed a detection limit of 1 μg/g and good assay reproducibility (RSD 8%), suitable for the simultaneous detection of the three targeted allergens. The developed methodology was tested with several commercially available foodstuffs, demonstrating its applicability. The results were in good agreement, in terms of sensitivity and reproducibility, with those obtained with ELISA, PCR-gel agarose electrophoresis, and RT-PCR.

Detection of Legionella pneumophila by real-time PCR for the mip gene.

PubMed

Wilson, Deborah A; Yen-Lieberman, Belinda; Reischl, Udo; Gordon, Steve M; Procop, Gary W

2003-07-01

A real-time PCR assay for the mip gene of Legionella pneumophila was tested with 27 isolates of L. pneumophila, 20 isolates of 14 other Legionella species, and 103 non-Legionella bacteria. Eight culture-positive and 40 culture-negative clinical specimens were tested. This assay was 100% sensitive and 100% specific for L. pneumophila.

Collaborative ring trial of the papaya endogenous reference gene and its polymerase chain reaction assays for genetically modified organism analysis.

PubMed

Wei, Jiaojun; Li, Feiwu; Guo, Jinchao; Li, Xiang; Xu, Junfeng; Wu, Gang; Zhang, Dabing; Yang, Litao

2013-11-27

The papaya (Carica papaya L.) Chymopapain (CHY) gene has been reported as a suitable endogenous reference gene for genetically modified (GM) papaya detection in previous studies. Herein, we further validated the use of the CHY gene and its qualitative and quantitative polymerase chain reaction (PCR) assays through an interlaboratory collaborative ring trial. A total of 12 laboratories working on detection of genetically modified organisms participated in the ring trial and returned test results. Statistical analysis of the returned results confirmed the species specificity, low heterogeneity, and single-copy number of the CHY gene among different papaya varieties. The limit of detection of the CHY qualitative PCR assay was 0.1%, while the limit of quantification of the quantitative PCR assay was ∼25 copies of haploid papaya genome with acceptable PCR efficiency and linearity. The differences between the tested and true values of papaya content in 10 blind samples ranged from 0.84 to 6.58%. These results indicated that the CHY gene was suitable as an endogenous reference gene for the identification and quantification of GM papaya.

A Multiplex Snapback Primer System for the Enrichment and Detection of JAK2 V617F and MPL W515L/K Mutations in Philadelphia-Negative Myeloproliferative Neoplasms

PubMed Central

Zhang, Yunqing; Zhang, Xinju; Xu, Xiao; Kang, Zhihua; Li, Shibao; Zhang, Chen; Su, Bing

2014-01-01

A multiplex snapback primer system was developed for the simultaneous detection of JAK2 V617F and MPL W515L/K mutations in Philadelphia chromosome- (Ph-) negative myeloproliferative neoplasms (MPNs). The multiplex system comprises two snapback versus limiting primer sets for JAK2 and MPL mutation enrichment and detection, respectively. Linear-After exponential (LATE) PCR strategy was employed for the primer design to maximize the amplification efficiency of the system. Low ionic strength buffer and rapid PCR protocol allowed for selective amplification of the mutant alleles. Amplification products were analyzed by melting curve analysis for mutation identification. The multiplex system archived 0.1% mutation load sensitivity and <5% coefficient of variation inter-/intra-assay reproducibility. 120 clinical samples were tested by the multiplex snapback primer assay, and verified with amplification refractory system (ARMS), quantitative PCR (qPCR) and Sanger sequencing method. The multiplex system, with a favored versatility, provided the molecular diagnosis of Ph-negative MPNs with a suitable implement and simplified the genetic test process. PMID:24729973

A multisite blinded study for the detection of BRAF mutations in formalin-fixed, paraffin-embedded malignant melanoma

PubMed Central

Richter, Anna; Grieu, Fabienne; Carrello, Amerigo; Amanuel, Benhur; Namdarian, Kateh; Rynska, Aleksandra; Lucas, Amanda; Michael, Victoria; Bell, Anthony; Fox, Stephen B.; Hewitt, Chelsee A.; Do, Hongdo; McArthur, Grant A.; Wong, Stephen Q.; Dobrovic, Alexander; Iacopetta, Barry

2013-01-01

Melanoma patients with BRAF mutations respond to treatment with vemurafenib, thus creating a need for accurate testing of BRAF mutation status. We carried out a blinded study to evaluate various BRAF mutation testing methodologies in the clinical setting. Formalin-fixed, paraffin-embedded melanoma samples were macrodissected before screening for mutations using Sanger sequencing, single-strand conformation analysis (SSCA), high resolution melting analysis (HRM) and competitive allele-specific TaqMan® PCR (CAST-PCR). Concordance of 100% was observed between the Sanger sequencing, SSCA and HRM techniques. CAST-PCR gave rapid and accurate results for the common V600E and V600K mutations, however additional assays are required to detect rarer BRAF mutation types found in 3–4% of melanomas. HRM and SSCA followed by Sanger sequencing are effective two-step strategies for the detection of BRAF mutations in the clinical setting. CAST-PCR was useful for samples with low tumour purity and may also be a cost-effective and robust method for routine diagnostics. PMID:23584600

The Applicability of TaqMan-Based Quantitative Real-Time PCR Assays for Detecting and Enumerating Cryptosporidium spp. Oocysts in the Environment

PubMed Central

Staggs, Sarah E.; Beckman, Erin M.; Keely, Scott P.; Mackwan, Reena; Ware, Michael W.; Moyer, Alan P.; Ferretti, James A.; Sayed, Abu; Xiao, Lihua; Villegas, Eric N.

2013-01-01

Quantitative real-time polymerase chain reaction (qPCR) assays to detect Cryptosporidium oocysts in clinical samples are increasingly being used to diagnose human cryptosporidiosis, but a parallel approach for detecting and identifying Cryptosporidium oocyst contamination in surface water sources has yet to be established for current drinking water quality monitoring practices. It has been proposed that Cryptosporidium qPCR-based assays could be used as viable alternatives to current microscopic-based detection methods to quantify levels of oocysts in drinking water sources; however, data on specificity, analytical sensitivity, and the ability to accurately quantify low levels of oocysts are limited. The purpose of this study was to provide a comprehensive evaluation of TaqMan-based qPCR assays, which were developed for either clinical or environmental investigations, for detecting Cryptosporidium oocyst contamination in water. Ten different qPCR assays, six previously published and four developed in this study were analyzed for specificity and analytical sensitivity. Specificity varied between all ten assays, and in one particular assay, which targeted the Cryptosporidium 18S rRNA gene, successfully detected all Cryptosporidium spp. tested, but also cross-amplified T. gondii, fungi, algae, and dinoflagellates. When evaluating the analytical sensitivity of these qPCR assays, results showed that eight of the assays could reliably detect ten flow-sorted oocysts in reagent water or environmental matrix. This study revealed that while a qPCR-based detection assay can be useful for detecting and differentiating different Cryptosporidium species in environmental samples, it cannot accurately measure low levels of oocysts that are typically found in drinking water sources. PMID:23805235