Science.gov

Sample records for nested-multiplex pcr detection

  1. A novel nested multiplex polymerase chain reaction (PCR) assay for differential detection of Entamoeba histolytica, E. moshkovskii and E. dispar DNA in stool samples

    PubMed Central

    Khairnar, Krishna; Parija, Subhash C

    2007-01-01

    Background E. histolytica, a pathogenic amoeba, is indistinguishable in its cyst and trophozoite stages from those of non-pathogenic E. moshkovskii and E. dispar by light microscopy. We have developed a nested multiplex PCR targeting a 16S-like rRNA gene for differential detection of all the three morphologically similar forms of E. histolytica, E. moshkovskii and E. dispar simultaneously in stool samples. Results The species specific product size for E. histolytica, E. moshkovskii and E. dispar was 439, 553 and 174 bp respectively, which was clearly different for all the three Entamoeba species. The nested multiplex PCR showed a sensitivity of 94% and specificity of 100% for the demonstration of E. histolytica, E. moshkovskii and E. dispar DNA in stool samples. The PCR was positive for E. histolytica, E. moshkovskii and E. dispar in a total of 190 out of 202 stool specimens (94% sensitive) that were positive for E. histolytica/E. dispar/E. moshkovskii by examination of stool by microscopy and/or culture. All the 35 negative control stool samples that were negative for E. histolytica/E. dispar/E. moshkovskii by microscopy and culture were also found negative by the nested multiplex PCR (100% specific). The result from the study shows that only 34.6% of the patient stool samples that were positive for E. histolytica/E. dispar/E. moshkovskii by examination of stool by microscopy and/or culture, were actually positive for pathogenic E. histolytica and the remaining majority of the stool samples were positive for non-pathogenic E. dispar or E. moshkovskii as demonstrated by the use of nested multiplex PCR. Conclusion The present study reports a new nested multiplex PCR strategy for species specific detection and differentiation of E. histolytica, E. dispar and E. moshkovskii DNA in stool specimens. The test is highly specific, sensitive and also rapid, providing the results within 12 hours of receiving stool specimens. PMID:17524135

  2. Comparison of nested-multiplex, Taqman & SYBR Green real-time PCR in diagnosis of amoebic liver abscess in a tertiary health care institute in India

    PubMed Central

    Dinoop, K.P.; Parija, Subhash Chandra; Mandal, Jharna; Swaminathan, R.P.; Narayanan, P.

    2016-01-01

    Background & objectives: Amoebiasis is a common parasitic infection caused by Entamoeba histolytica and amoebic liver abscess (ALA) is the most common extraintestinal manifestation of amoebiasis. The aim of this study was to standardise real-time PCR assays (Taqman and SYBR Green) to detect E. histolytica from liver abscess pus and stool samples and compare its results with nested-multiplex PCR. Methods: Liver abscess pus specimens were subjected to DNA extraction. The extracted DNA samples were subjected to amplification by nested-multiplex PCR, Taqman (18S rRNA) and SYBR Green real-time PCR (16S-like rRNA assays to detect E. histolytica/E. dispar/E. moshkovskii). The amplification products were further confirmed by DNA sequence analysis. Receiver operator characteristic (ROC) curve analysis was done for nested-multiplex and SYBR Green real-time PCR and the area under the curve was calculated for evaluating the accuracy of the tests to dignose ALA. Results: In all, 17, 19 and 25 liver abscess samples were positive for E. histolytica by nested-multiplex PCR, SYBR Green and Taqman real-time PCR assays, respectively. Significant differences in detection of E. histolytica were noted in the real-time PCR assays evaluated (P<0.0001). The nested-multiplex PCR, SYBR Green real-time PCR and Taqman real-time PCR evaluated showed a positivity rate of 34, 38 and 50 per cent, respectively. Based on ROC curve analysis (considering Taqman real-time PCR as the gold standard), it was observed that SYBR Green real-time PCR was better than conventional nested-multiplex PCR for the diagnosis of ALA. Interpretation & conclusions: Taqman real-time PCR targeting the 18S rRNA had the highest positivity rate evaluated in this study. Both nested multiplex and SYBR Green real-time PCR assays utilized were evaluated to give accurate results. Real-time PCR assays can be used as the gold standard in rapid and reliable diagnosis, and appropriate management of amoebiasis, replacing the

  3. Detection and Typing of Human Papilloma Viruses by Nested Multiplex Polymerase Chain Reaction Assay in Cervical Cancer

    PubMed Central

    Jalal Kiani, Seyed; Shatizadeh Malekshahi, Somayeh; Yousefi Ghalejoogh, Zohreh; Ghavvami, Nastaran; Shafiei Jandaghi, Nazanin Zahra; Shahsiah, Reza; Jahanzad, Isa; Yavarian, Jila

    2015-01-01

    Background: Cervical cancer is the leading cause of death from cancer in under-developed countries. Human papilloma virus (HPV) 16 and 18 are the most prevalent types associated with carcinogenesis in the cervix. Conventional Polymerase Chain Reaction (PCR), type-specific and consensus primer-based PCR followed by sequencing, Restriction Fragment Length Polymorphism (RFLP) or hybridization by specific probes are common methods for HPV detection and typing. In addition, some researchers have developed a multiplex PCR for simultaneous detection and typing of different HPVs. Objectives: The aim of the present study was to investigate the prevalence of HPV infection and its types in cervical Squamous Cell Carcinoma (SCC) using the Nested Multiplex PCR (NMPCR) assay. Patients and Methods: Sixty-six samples with histologically confirmed SCC were evaluated. Total DNA was isolated by phenol–chloroform extraction and ethanol precipitation. Nested multiplex PCR was performed with first-round PCR by GP-E6/E7 consensus primers for amplification of the genomic DNA of all known mucosal HPV genotypes and second-round PCR by type-specific multiplex PCR primer cocktails. Results: Human papilloma virus infection was detected in 78.8% of samples, with the highest prevalence of HPV 16 (60.6%) while concurrent infections with two types was detected in 10.6%. Conclusions: The NMPCR assay is more convenient and easy for analysis of results, which is important for fast diagnosis and patient management, in a type-specific manner. PMID:26865940

  4. Nested multiplex PCR--a feasible technique to study partial community of arbuscular mycorrhizal fungi in field-growing plant root.

    PubMed

    Dong, Xiuli; Zhao, Bin

    2006-08-01

    Plant can be infected by different arbuscular mycorrhizal fungi, but little is known about the interaction between them within root tissues mainly because different species cannot be distinguished on the basis of fungal structure. Accurate species identification of Arbuscular mycorrhizal fungi (AMF) colonized in plant roots is the cornerstone of mycorrhizal study, yet this fundamental step is impossible through its morphological character alone. For accurate, rapid and inexpensive detection of partial mycorrhizal fungal community in plant roots, a nested multiplex polymerase chain reaction (PCR) was developed in this study. Five discriminating primers designed based on the variable region of the 5' end of the large ribosomal subunit were used in the experiment for testing their specificity and the sensitivity in nested PCR by using spores from Glomus mosseae (BEG12), Glomus intraradices (BEG141), Scutellospora castaneae (BEG1) and two unidentified Glomus sp. HAUO3 and HAUO4. The feasibility assay of nested multiplex PCR was conducted by use of spore mixture, Astragalus sinicum roots co-inoculated with 4 species of arbuscular mycorrhizal fungi from pot cultures and 15 different field-growing plant roots respectively after analyses of the compatibility of primers. The result indicated that the sensitivity was in the same range as that of the corresponding single PCR reaction. Overall accuracy was 95%. The efficiency and sensitivity of this multiplex PCR procedure provided a rapid and easy way to simultaneously detect several of arbuscular mycorrhiza fungal species in a same plant root system. PMID:16989281

  5. Identification of root rot fungi in nursery seedlings by nested multiplex PCR.

    PubMed Central

    Hamelin, R C; Bérubé, P; Gignac, M; Bourassa, M

    1996-01-01

    The internal transcribed spacer (ITS) of the ribosomal DNA (rDNA) subunit repeat was sequenced in 12 isolates of Cylindrocladium floridanum and 11 isolates of Cylindrocarpon destructans. Sequences were aligned and compared with ITS sequences of other fungi in GenBank. Some intraspecific variability was present within our collections of C. destructans but not in C. floridanum. Three ITS variants were identified within C. destructans, but there was no apparent association between ITS variants and host or geographic origin. Two internal primers were synthesized for the specific amplification of portions of the ITS for C. floridanum, and two primers were designed to amplify all three variants of C. destructans. The species-specific primers amplified PCR products of the expected length when tested with cultures of C, destructans and C. floridanum from white spruce, black spruce, Norway spruce, red spruce, jack pine, red pine, and black walnut from eight nurseries and three plantations in Quebec. No amplification resulted from PCR reactions on fungal DNA from 26 common contaminants of conifer roots. For amplifications directly from infected tissues, a nested primer PCR using two rounds of amplification was combined with multiplex PCR approach resulting in the amplification of two different species-specific PCR fragments in the same reaction. First, the entire ITS was amplified with one universal primer and a second primer specific to fungi; a second round of amplification was carried out with species-specific primers that amplified a 400-bp PCR product from C. destructans and a 328-bp product from C. floridanum. The species-specific fragments were amplified directly from infected roots from which one or the two fungi had been isolated. PMID:8899993

  6. Digital PCR for detection of citrus pathogens

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Citrus trees are often infected with multiple pathogens of economic importance, especially those with insect or mite vectors. Real-time/quantitative PCR (qPCR) has been used for high-throughput detection and relative quantification of pathogens; however, target reference or standards are required. I...

  7. Transgene Detection by Digital Droplet PCR

    PubMed Central

    Moser, Dirk A.; Braga, Luca; Raso, Andrea; Zacchigna, Serena; Giacca, Mauro; Simon, Perikles

    2014-01-01

    Somatic gene therapy is a promising tool for the treatment of severe diseases. Because of its abuse potential for performance enhancement in sports, the World Anti-Doping Agency (WADA) included the term ‘gene doping’ in the official list of banned substances and methods in 2004. Several nested PCR or qPCR-based strategies have been proposed that aim at detecting long-term presence of transgene in blood, but these strategies are hampered by technical limitations. We developed a digital droplet PCR (ddPCR) protocol for Insulin-Like Growth Factor 1 (IGF1) detection and demonstrated its applicability monitoring 6 mice injected into skeletal muscle with AAV9-IGF1 elements and 2 controls over a 33-day period. A duplex ddPCR protocol for simultaneous detection of Insulin-Like Growth Factor 1 (IGF1) and Erythropoietin (EPO) transgenic elements was created. A new DNA extraction procedure with target-orientated usage of restriction enzymes including on-column DNA-digestion was established. In vivo data revealed that IGF1 transgenic elements could be reliably detected for a 33-day period in DNA extracted from whole blood. In vitro data indicated feasibility of IGF1 and EPO detection by duplex ddPCR with high reliability and sensitivity. On-column DNA-digestion allowed for significantly improved target detection in downstream PCR-based approaches. As ddPCR provides absolute quantification, it ensures excellent day-to-day reproducibility. Therefore, we expect this technique to be used in diagnosing and monitoring of viral and bacterial infection, in detecting mutated DNA sequences as well as profiling for the presence of foreign genetic material in elite athletes in the future. PMID:25375130

  8. Propidium monoazide reverse transcriptase PCR and RT-qPCR for detecting infectious enterovirus and norovirus.

    PubMed

    Karim, Mohammad R; Fout, G Shay; Johnson, Clifford H; White, Karen M; Parshionikar, Sandhya U

    2015-07-01

    Presently there is no established cell line or small animal model that allows for the detection of infectious human norovirus. Current methods based on RT-PCR and RT-qPCR detect both infectious and non-infectious virus and thus the conclusions that may be drawn regarding the public health significance of positive findings are limited. In this study, PMA RT-PCR and RT-qPCR assays were evaluated for selective detection of infectious poliovirus, murine norovirus (MNV-1), and Norwalk virus. Viruses were inactivated using heat, chlorine, and ultraviolet light (UV). Infectious and non-infectious viruses were treated with PMA before RT-PCR and RT-qPCR. PMA RT-PCR was able to differentiate selectively between infectious and heat and chlorine inactivated poliovirus. PMA RT-PCR was able to differentiate selectively between infectious and noninfectious murine norovirus only when inactivated by chlorine. However, PMA RT-PCR could not differentiate infectious Norwalk virus from virus particles rendered non-infectious by any treatment. PMA RT-PCR assay was not able to differentiate between infectious and UV inactivated viruses suggesting that viral capsid damage may be necessary for PMA to enter and bind to the viral genome. PMA RT-PCR on naked MNV-1 and Norwalk virus RNA suggest that PMA RT-PCR can be used to detect intact, potentially infectious MNV-1 and Norwalk viruses and can be used to exclude the detection of free viral RNA by PCR assay. PMID:25796356

  9. First case of detection of Plasmodium knowlesi in Spain by Real Time PCR in a traveller from Southeast Asia.

    PubMed

    Ta, Tang Thuy-Huong; Salas, Ana; Ali-Tammam, Marwa; Martínez, María Del Carmen; Lanza, Marta; Arroyo, Eduardo; Rubio, Jose Miguel

    2010-01-01

    Previously, Plasmodium knowlesi was not considered as a species of Plasmodium that could cause malaria in human beings, as it is parasite of long-tailed (Macaca fascicularis) and pig-tailed (Macaca nemestrina) macaques found in Southeast Asia. A case of infection by P. knowlesi is described in a Spanish traveller, who came back to Spain with daily fever after his last overseas travel, which was a six-month holiday in forested areas of Southeast Asia between 2008 and 2009. His P. knowlesi infection was detected by multiplex Real time quantitative PCR and confirmed by sequencing the amplified fragment. Using nested multiplex malaria PCR (reference method in Spain) and a rapid diagnostic test, the P. knowlesi infection was negative. This patient was discharged and asymptomatic when the positive result to P. knowlesi was reported. Prior to this case, there have been two more reports of European travellers with malaria caused by P. knowlesi, a Finnish man who travelled to Peninsular Malaysia during four weeks in March 2007, and a Swedish man who did a short visit to Malaysian Borneo in October 2006. Taken together with this report of P. knowlesi infection in a Spanish traveller returning from Southeast Asia, this is the third case of P. knowlesi infection in Europe, indicating that this simian parasite can infect visitors to endemic areas in Southeast Asia. This last European case is quite surprising, given that it is an untreated-symptomatic P. knowlesi in human, in contrast to what is currently known about P. knowlesi infection. Most previous reports of human P. knowlesi malaria infections were in adults, often with symptoms and relatively high parasite densities, up to the recent report in Ninh Thuan province, located in the southern part of central Vietnam, inhabited mainly by the Ra-glai ethnic minority, in which all P. knowlesi infections were asymptomatic, co-infected with P. malariae, with low parasite densities and two of the three identified cases were very

  10. Propidium monoazide reverse transcription PCR and RT-qPCR for detecting infectious enterovirus and norovirus

    EPA Science Inventory

    Presently there is no established cell line or small animal model that allows for the detection of infectious human norovirus. Current methods based on RT-PCR and RT-qPCR detect both infectious and non-infectious virus and thus the conclusions that may be drawn regarding the publ...

  11. Detection of virulence genes of Clostridium difficile by multiplex PCR.

    PubMed

    Antikainen, Jenni; Pasanen, Tanja; Mero, Sointu; Tarkka, Eveliina; Kirveskari, Juha; Kotila, Saara; Mentula, Silja; Könönen, Eija; Virolainen-Julkunen, Anni-Riitta; Vaara, Martti; Tissari, Päivi

    2009-08-01

    Clostridium difficile strains belonging to the PCR ribotype 027, pulse-field gel electrophoresis (PFGE) type NAP1, toxinotype III and restriction endonuclease analysis group BI harbouring mutations in the tcdC gene and possessing binary toxin components A and B have been described to cause epidemics with increased morbidity and mortality. In the present study we developed a conventional multiplex PCR designed to detect selected virulence associated markers of the hypervirulent C. difficile PCR ribotype 027. The multiplex PCR assay detected the major toxins A and B, binary toxin components A and B as well as a possible deletion in the tcdC gene: a characteristic pattern of amplification products for the PCR ribotype 027 strains was detected. This rather simple method was specific for the screening of this hypervirulent C. difficile strain. The correlation between the multiplex PCR and PCR ribotyping methods was excellent. The sensitivity and specificity were 100% in our epidemiological situation. In conclusion, this multiplex PCR was found useful in the preliminary screening for the hypervirulent C. difficile PCR ribotype 027. PMID:19664132

  12. IDH1 mutation detection by droplet digital PCR in glioma.

    PubMed

    Wang, Jing; Zhao, Yi-ying; Li, Jian-feng; Guo, Cheng-cheng; Chen, Fu-rong; Su, Hong-kai; Zhao, Hua-fu; Long, Ya-kang; Shao, Jian-yong; To, Shing shun Tony; Chen, Zhong-ping

    2015-11-24

    Glioma is the most frequent central nervous system tumor in adults. The overall survival of glioma patients is disappointing, mostly due to the poor prognosis of glioblastoma (Grade IV glioma). Isocitrate dehydrogenase (IDH) is a key factor in metabolism and catalyzes the oxidative decarboxylation of isocitrate. Mutations in IDH genes are observed in over 70% of low-grade gliomas and some cases of glioblastoma. As the most frequent mutation, IDH1(R132H) has been served as a predictive marker of glioma patients. The recently developed droplet digital PCR (ddPCR) technique generates a large amount of nanoliter-sized droplets, each of which carries out a PCR reaction on one template. Therefore, ddPCR provides high precision and absolute quantification of the nucleic acid target, with wide applications for both research and clinical diagnosis. In the current study, we collected 62 glioma tissue samples (Grade II to IV) and detected IDH1 mutations by Sanger direct sequencing, ddPCR, and quantitative real-time PCR (qRT-PCR). With the results from Sanger direct sequencing as the standard, the characteristics of ddPCR were compared with qRT-PCR. The data indicated that ddPCR was much more sensitive and much easier to interpret than qRT-PCR. Thus, we demonstrated that ddPCR is a reliable and sensitive method for screening the IDH mutation. Therefore, ddPCR is able to applied clinically in predicting patient prognosis and selecting effective therapeutic strategies. Our data also supported that the prognosis of Grade II and III glioma was better in patients with an IDH mutation than in those without mutation. PMID:26485760

  13. IDH1 mutation detection by droplet digital PCR in glioma

    PubMed Central

    Wang, Jing; Zhao, Yi-ying; Li, Jian-feng; Guo, Cheng-cheng; Chen, Fu-rong; Su, Hong-kai; Zhao, Hua-fu; Long, Ya-kang; Shao, Jian-yong; Tony To, Shing-shun; Chen, Zhong-ping

    2015-01-01

    Glioma is the most frequent central nervous system tumor in adults. The overall survival of glioma patients is disappointing, mostly due to the poor prognosis of glioblastoma (Grade IV glioma). Isocitrate dehydrogenase (IDH) is a key factor in metabolism and catalyzes the oxidative decarboxylation of isocitrate. Mutations in IDH genes are observed in over 70% of low-grade gliomas and some cases of glioblastoma. As the most frequent mutation, IDH1(R132H) has been served as a predictive marker of glioma patients. The recently developed droplet digital PCR (ddPCR) technique generates a large amount of nanoliter-sized droplets, each of which carries out a PCR reaction on one template. Therefore, ddPCR provides high precision and absolute quantification of the nucleic acid target, with wide applications for both research and clinical diagnosis. In the current study, we collected 62 glioma tissue samples (Grade II to IV) and detected IDH1 mutations by Sanger direct sequencing, ddPCR, and quantitative real-time PCR (qRT-PCR). With the results from Sanger direct sequencing as the standard, the characteristics of ddPCR were compared with qRT-PCR. The data indicated that ddPCR was much more sensitive and much easier to interpret than qRT-PCR. Thus, we demonstrated that ddPCR is a reliable and sensitive method for screening the IDH mutation. Therefore, ddPCR is able to applied clinically in predicting patient prognosis and selecting effective therapeutic strategies. Our data also supported that the prognosis of Grade II and III glioma was better in patients with an IDH mutation than in those without mutation. PMID:26485760

  14. PCR assays for detection of Baylisascaris procyonis eggs and larvae.

    PubMed

    Dangoudoubiyam, Sriveny; Vemulapalli, Ramesh; Kazacos, Kevin R

    2009-06-01

    The objective of this study was to develop polymerase chain reaction (PCR) assays for detection of Baylisascaris procyonis eggs and larvae in fecal, environmental, and tissue samples. We have optimized conventional and real-time PCR assays for B. procyonis using the mitochondrial cytochrome oxidase 2 gene as the target for amplification. The lower limit of detection of the parasite genomic DNA was 10 pg in the conventional PCR and 100 fg in the real-time PCR. In both PCR assays, specific amplification of a 146 bp product was achieved with DNA extracted from a single in vitro hatched B. procyonis larva and also from canine fecal samples spiked with as few as 20 unembryonated B. procyonis eggs per gram of feces. The PCR assays were successfully used for detection of B. procyonis eggs and larvae in fecal, environmental, and tissue samples. No DNA amplification was seen when the genomic DNA of related ascarids (including B. transfuga) and a hookworm was used as template in the PCR; however, amplification was seen with the very closely related B. columnaris. PMID:19090651

  15. Simultaneous Detection of Ricin and Abrin DNA by Real-Time PCR (qPCR)

    PubMed Central

    Felder, Eva; Mossbrugger, Ilona; Lange, Mirko; Wölfel, Roman

    2012-01-01

    Ricin and abrin are two of the most potent plant toxins known and may be easily obtained in high yield from the seeds using rather simple technology. As a result, both toxins are potent and available toxins for criminal or terrorist acts. However, as the production of highly purified ricin or abrin requires sophisticated equipment and knowledge, it may be more likely that crude extracts would be used by non-governmental perpetrators. Remaining plant-specific nucleic acids in these extracts allow the application of a real-time PCR (qPCR) assay for the detection and identification of abrin or ricin genomic material. Therefore, we have developed a duplex real-time PCR assays for simultaneous detection of ricin and abrin DNA based on the OmniMix HS bead PCR reagent mixture. Novel primers and hybridization probes were designed for detection on a SmartCycler instrument by using 5′-nuclease technology. The assay was thoroughly optimized and validated in terms of analytical sensitivity. Evaluation of the assay sensitivity by probit analysis demonstrated a 95% probability of detection at 3 genomes per reaction for ricin DNA and 1.2 genomes per reaction for abrin DNA. The suitability of the assays was exemplified by detection of ricin and abrin contaminations in a food matrix. PMID:23105972

  16. Quantitative Detection of Spiroplasma Citri by Real Time PCR

    Technology Transfer Automated Retrieval System (TEKTRAN)

    There is a need to develop an accurate and rapid method to detect Spiroplasma citri, the causal agent of citrus stubborn disease for use in epidemiology studies. Quantitative real-time PCR was developed for detection of S. citri. Two sets of primers based on sequences from the P58 putative adhesin ...

  17. UTILIZATION OF PCR TO DETECT SALMONELLA ON TURKEY CARCASSES

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The risk which is presented by food-borne pathogens to the consumer demonstrates the need to utilize rapid methods for the detection of these microbes. This study compared conventional microbiology with the application of PCR assays to detect Salmonella on turkey carcasses at a processing plant in ...

  18. Detection of Treponema pallidum in the vitreous by PCR

    PubMed Central

    Müller, M; Ewert, I; Hansmann, F; Tiemann, C; Hagedorn, H J; Solbach, W; Roider, J; Nölle, B; Laqua, H; Hoerauf, H

    2007-01-01

    Background Ocular involvement of syphilis still poses a clinical challenge due to the chameleonic behaviour of the disease. As the serodiagnosis has significant limitations, the direct detection of Treponema pallidum (TP) in the vitreous represents a desirable diagnostic tool. Methods Real‐time polymerase chain reaction (PCR) for the detection of TP was applied in diagnostic vitrectomies of two patients with acute chorioretinitis. Qualitative verification of TP by real‐time PCR and melting point analysis according to a modified protocol was ruled out. Patients underwent complete ophthalmological examination with fundus photographs, fluorescein angiography, serological examination, antibiotic treatment and follow‐up. Results In two cases of acute chorioretinitis of unknown origin, real‐time PCR of vitreous specimens of both patients provided evidence of TP and was 100% specific. Initial diagnosis of presumed viral retinitis was ruled out by PCR of vitreous specimen. Patients were treated with systemic antibiotics and showed prompt improvement in visual function and resolution of fundus lesions. Conclusions With real‐time PCR, detection of TP in the vitreous was possible and delivered a sensitive, quick and inexpensive answer to a disease rather difficult to assess. In cases of acute chorioretinitis, the use of PCR‐based assays of vitreous specimens in the diagnostic evaluation of patients is advisable. Although syphilitic chorioretinitis is a rare disease, PCR should include search for TP, as diagnostic dilemmas prolong definitive treatment in a sight‐threatening disease. PMID:17108014

  19. Sensitive detection of sample interference in environmental qPCR.

    PubMed

    Green, Hyatt C; Field, Katharine G

    2012-06-15

    Sample interference in environmental applications of quantitative PCR (qPCR) can prevent accurate estimations of molecular markers in the environment. We developed a spike-and-recovery approach using a mutant strain of Escherichia coli that contains a chromosomal insertion of a mutant GFP gene. The method was tested in water samples by separately reducing extraction efficiency or adding humic acids and ethanol, compounds that often contaminate environmental DNA extracts, and analyzing qPCR amplification of the spiked E. coli control and human fecal Bacteroides markers (HF183 and HF134). This approach, coupled with previously developed kinetic outlier detection (KOD) methods, allowed sensitive detection of PCR inhibition at much lower inhibitor concentrations than alternative approaches using Cq values or amplification efficiencies. Although HF183 was more sensitive to the effects of qPCR inhibitors than the E. coli control assay, KOD methods correctly identified inhibition of both control and HF183 assays in samples containing as little as 0.1 ng humic acids per reaction or 5% ethanol. Because sigmoidal modeling methods allow distinction of qPCR inhibition from poor DNA recovery, we were able to simultaneously identify qPCR-inhibited reactions and estimate recovery of nucleic acids in environmental samples using a single control assay. Since qPCR is currently used to estimate important water quality parameters that have serious economic and human health outcomes, these results are timely. While we demonstrate the methods in the context of water quality regulation, they will be useful in all areas of environmental research that use qPCR. PMID:22560896

  20. Detection of Treponema pallidum by a sensitive reverse transcriptase PCR.

    PubMed Central

    Centurion-Lara, A; Castro, C; Shaffer, J M; Van Voorhis, W C; Marra, C M; Lukehart, S A

    1997-01-01

    Syphilis is diagnosed by serologic testing or by identification of the causative agent, Treponema pallidum. The bacterium has historically been detected in clinical specimens by dark-field microscopy, immunostaining with polyclonal or monoclonal antibodies, or the rabbit inoculation test (RIT). RIT is considered to be very sensitive and specific, although it is available only in research settings and is not clinically useful due to the length of time required to obtain a result. In recent years, several PCR methods have been developed for the detection of T. pallidum, but none of these has shown a clear advantage in sensitivity over RIT. We have developed a specific and highly sensitive reverse transcriptase PCR (RT-PCR) that targets a 366 bp region of the 16S rRNA of T. pallidum. This RT-PCR can detect a single organism by Southern analysis when whole organisms are diluted and 10(-2) to 10(-3) T. pallidum organisms when RNA equivalents are used to make cDNA. The test was demonstrated to detect 10(-2) T. pallidum RNA equivalents in cerebrospinal fluid. Twenty different strains of T. pallidum, isolated from cerebrospinal fluids, aqueous humor, blood, and chancres, were shown to be detectable by this test. This efficient and sensitive technique could be more useful than existing methods for detecting very low numbers of organisms in clinical samples. PMID:9163442

  1. Ultrasensitive Antibody Detection by Agglutination-PCR (ADAP)

    PubMed Central

    2016-01-01

    Antibodies are widely used biomarkers for the diagnosis of many diseases. Assays based on solid-phase immobilization of antigens comprise the majority of clinical platforms for antibody detection, but can be undermined by antigen denaturation and epitope masking. These technological hurdles are especially troublesome in detecting antibodies that bind nonlinear or conformational epitopes, such as anti-insulin antibodies in type 1 diabetes patients and anti-thyroglobulin antibodies associated with thyroid cancers. Radioimmunoassay remains the gold standard for these challenging antibody biomarkers, but the limited multiplexability and reliance on hazardous radioactive reagents have prevented their use outside specialized testing facilities. Here we present an ultrasensitive solution-phase method for detecting antibodies, termed antibody detection by agglutination-PCR (ADAP). Antibodies bind to and agglutinate synthetic antigen–DNA conjugates, enabling ligation of the DNA strands and subsequent quantification by qPCR. ADAP detects zepto- to attomoles of antibodies in 2 μL of sample with a dynamic range spanning 5–6 orders of magnitude. Using ADAP, we detected anti-thyroglobulin autoantibodies from human patient plasma with a 1000-fold increased sensitivity over an FDA-approved radioimmunoassay. Finally, we demonstrate the multiplexability of ADAP by simultaneously detecting multiple antibodies in one experiment. ADAP’s combination of simplicity, sensitivity, broad dynamic range, multiplexability, and use of standard PCR protocols creates new opportunities for the discovery and detection of antibody biomarkers. PMID:27064772

  2. Comparison of Droplet Digital PCR and Quantitative PCR Assays for Quantitative Detection of Xanthomonas citri Subsp. citri.

    PubMed

    Zhao, Yun; Xia, Qingyan; Yin, Youping; Wang, Zhongkang

    2016-01-01

    Droplet digital polymerase chain reaction (ddPCR) is a novel molecular biology technique providing absolute quantification of target nucleic acids without the need for an external calibrator. Despite its emerging applications in medical diagnosis, there are few reports of its use for the detection of plant pathogens. This work was designed to assess the diagnosis potential of the ddPCR for absolute quantitative detection of Xanthomonas citri subsp. citri, a quarantine plant pathogenic bacterium that causes citrus bacterial canker in susceptible Citrus species. We transferred an established quantitative PCR (qPCR) assay for citrus bacterial canker diagnosis directly to the ddPCR format and compared the performance of the two methods. The qPCR assay has a broader dynamic range compared to the ddPCR assay and the ddPCR assay has a significantly higher degree of sensitivity compared to the qPCR assay. The influence of PCR inhibitors can be reduced considerably in the ddPCR assay because the collection of end-point fluorescent signals and the counting of binomial events (positive or negative droplets) are associated with a Poisson algorithm. The ddPCR assay also shows lower coefficient of variation compared to the qPCR assay especially in low target concentration. The linear association of the measurements by ddPCR and qPCR assays is strong (Pearson correlation = 0.8633; P<0.001). Receiver operating characteristic analysis indicates the ddPCR methodology is a more robust approach for diagnosis of citrus bacterial canker. In summary, the results demonstrated that the ddPCR assay has the potential for the quantitative detection of X. citri subsp. citri with high precision and accuracy as compared with the results from qPCR assay. Further studies are required to evaluate and validate the value of ddPCR technology in the diagnosis of plant disease and quarantine applications. PMID:27427975

  3. Comparison of Droplet Digital PCR and Quantitative PCR Assays for Quantitative Detection of Xanthomonas citri Subsp. citri

    PubMed Central

    Yin, Youping; Wang, Zhongkang

    2016-01-01

    Droplet digital polymerase chain reaction (ddPCR) is a novel molecular biology technique providing absolute quantification of target nucleic acids without the need for an external calibrator. Despite its emerging applications in medical diagnosis, there are few reports of its use for the detection of plant pathogens. This work was designed to assess the diagnosis potential of the ddPCR for absolute quantitative detection of Xanthomonas citri subsp. citri, a quarantine plant pathogenic bacterium that causes citrus bacterial canker in susceptible Citrus species. We transferred an established quantitative PCR (qPCR) assay for citrus bacterial canker diagnosis directly to the ddPCR format and compared the performance of the two methods. The qPCR assay has a broader dynamic range compared to the ddPCR assay and the ddPCR assay has a significantly higher degree of sensitivity compared to the qPCR assay. The influence of PCR inhibitors can be reduced considerably in the ddPCR assay because the collection of end-point fluorescent signals and the counting of binomial events (positive or negative droplets) are associated with a Poisson algorithm. The ddPCR assay also shows lower coefficient of variation compared to the qPCR assay especially in low target concentration. The linear association of the measurements by ddPCR and qPCR assays is strong (Pearson correlation = 0.8633; P<0.001). Receiver operating characteristic analysis indicates the ddPCR methodology is a more robust approach for diagnosis of citrus bacterial canker. In summary, the results demonstrated that the ddPCR assay has the potential for the quantitative detection of X. citri subsp. citri with high precision and accuracy as compared with the results from qPCR assay. Further studies are required to evaluate and validate the value of ddPCR technology in the diagnosis of plant disease and quarantine applications. PMID:27427975

  4. Simultaneous multiplex PCR detection of seven cucurbit-infecting viruses.

    PubMed

    Kwon, Ji Yeon; Hong, Jin Sung; Kim, Min Jea; Choi, Sun Hee; Min, Byeong Eun; Song, Eun Gyeong; Kim, Hyun Hee; Ryu, Ki Hyun

    2014-09-01

    Two multiplex polymerase chain reaction (PCR) systems using dual priming oligonucleotide (DPO) primers were developed for the simultaneous detection of seven cucurbit-infecting viruses. One system allows for the detection of papaya ringspot virus, watermelon mosaic virus, and zucchini yellow mosaic virus, whereas the other permits the detection of cucumber green mottle mosaic virus, cucumber fruit mottle mosaic virus, kyuri green mottle mosaic virus, and zucchini green mottle mosaic virus. Viral species-specific DPO primers developed in this study detected as little as 10 fg/μl of viral RNA under monoplex conditions and 10 pg/μl of viral RNA under multiplex conditions. Multiplex PCR using the DPO primer sets was capable of amplifying viral genes at annealing temperatures ranging from 53 °C to 63 °C. Whereas the use of conventional primers gave rise to non-specific bands, the DPO primers detected target viral genes in the absence of non-specific amplification. When these DPO multiplex primer sets were applied to virus-infected cucurbit samples obtained in the field, multiple infection as well as single infection was accurately identified. This novel approach could also detect multiple viruses in infected seeds. The reliability of multiplex PCR systems using DPO primers for plant virus detection is discussed. PMID:24937806

  5. DETECTION OF FECAL ENTEROCOCCI USING A REAL TIME PCR METHOD

    EPA Science Inventory

    In spite of their importance in public health, the detection of fecal enterococci is performed via culturing methods that are time consuming and that are subject to inaccuracies that relate to their culturable status. In order to address these problems, a real time PCR (TaqMan) ...

  6. PCR detection of Helicobacter pylori in clinical samples.

    PubMed

    Rimbara, Emiko; Sasatsu, Masanori; Graham, David Y

    2013-01-01

    Helicobacter pylori is an important pathogen whose primary niche is the human stomach. H. pylori is etiologically associated with gastric inflammation (gastritis), peptic ulcer disease, and gastric cancer. Both noninvasive (e.g., urea breath and stool antigen tests) and invasive (gastric biopsy for histology, culture, or PCR) tests are used for diagnosis. PCR detection of H. pylori has been reported using a variety of clinical samples including gastric biopsy, gastric juice, saliva, dental plaque, and stools as well as environmental samples. Whenever possibly, noninvasive tests are preferred over invasive tests. H. pylori are excreted in the stool. Culture from stool is variable whereas stool antigen testing is widely used. Stool consists of a complicated mixture of commensal bacteria and chemicals and often includes inhibitors of PCR. Nevertheless, simple extraction methods are available to efficiently extract DNA from human stools and nested-PCR targeting the 23S rRNA gene have proven to be highly sensitive for the detection of H. pylori. Detection of clarithromycin susceptibility/resistance is important clinically and the mutation of the 23S rRNA gene responsible for resistance can also be detected using stool. This described method can be modified for other clinical samples such as gastric juice or biopsy material. PMID:23104297

  7. Detection of carbapenemases in Enterobacteriaceae by a commercial multiplex PCR.

    PubMed

    Kaase, Martin; Szabados, Florian; Wassill, Lars; Gatermann, Sören G

    2012-09-01

    A commercial multiplex PCR (hyplex SuperBug ID) was tested with a collection of 132 clinical Enterobacteriaceae strains producing different carbapenemases. The sensitivity for the detection of KPC-, VIM-, NDM-, and OXA-48-encoding genes was 100%, whereas two IMP variants were missed. PMID:22785190

  8. Detection of viable Cryptosporidium parvum oocysts by PCR.

    PubMed Central

    Wagner-Wiening, C; Kimmig, P

    1995-01-01

    PCR was used to detect and specifically identify a gene fragment from Cryptosporidium parvum. An 873-bp region of a 2,359-bp DNA fragment encoding a repetitive oocyst protein of C. parvum was shown to be specifically amplified in C. parvum. An excystation protocol before DNA extraction allowed the differentiation between live and dead Cryptosporidium parvum oocysts. PMID:8534121

  9. 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

  10. Electrochemiluminescence-PCR detection of genetically modified organisms

    NASA Astrophysics Data System (ADS)

    Liu, Jinfeng; Xing, Da; Shen, Xingyan; Zhu, Debin

    2005-01-01

    The detection methods for genetically modified (GM) components in foods have been developed recently. But many of them are complicated and time-consuming; some of them need to use the carcinogenic substance, and can"t avoid false-positive results. In this study, an electrochemiluminescence polymerase chain reaction (ECL-PCR) method for detection GM tobaccos is proposed. The Cauliflower mosaic virus 35S (CaMV35S) promoter was amplified by PCR, Then hybridized with a Ru(bpy)32+ (TBR)-labeled and a biotinylated probe. The hybridization products were captured onto streptavidin-coated paramagnetic beads, and detected by measuring the electrochemiluminescence (ECL) signal of the TBR label. Whether the tobaccos contain GM components was discriminated by detecting the ECL signal of CaMV35S promoter. The experiment results show that the detection limit for CaMV35S promoter is 100 fmol, and the GM components can be clearly identified in GM tobaccos. The ECL-PCR method provide a new means in GMOs detection due to its safety, simplicity and high efficiency.

  11. Development of a PCR Assay for Rapid Detection of Enterococci

    PubMed Central

    Ke, Danbing; Picard, François J.; Martineau, Francis; Ménard, Christian; Roy, Paul H.; Ouellette, Marc; Bergeron, Michel G.

    1999-01-01

    Enterococci are becoming major nosocomial pathogens, and increasing resistance to vancomycin has been well documented. Conventional identification methods, which are based on culturing, require 2 to 3 days to provide results. PCR has provided a means for the culture-independent detection of enterococci in a variety of clinical specimens and is capable of yielding results in just a few hours. However, all PCR-based assays developed so far are species specific only for clinically important enterococci. We have developed a PCR-based assay which allows the detection of enterococci at the genus level by targeting the tuf gene, which encodes elongation factor EF-Tu. Initially, we compared the nucleotide sequences of the tuf gene from several bacterial species (available in public databases) and designed degenerate PCR primers derived from conserved regions. These primers were used to amplify a target region of 803 bp from four enterococcal species (Enterococcus avium, E. faecalis, E. faecium, and E. gallinarum). Subsequently, the complete nucleotide sequences of these amplicons were determined. The analysis of a multiple alignment of these sequences revealed regions conserved among enterococci but distinct from those of other bacteria. PCR primers complementary to these regions allowed amplification of genomic DNAs from 14 of 15 species of enterococci tested (E. solitarius DNA could not be amplified). There was no amplification with a majority of 79 nonenterococcal bacterial species, except for 2 Abiotrophia species and several Listeria species. Furthermore, this assay efficiently amplified all 159 clinical isolates of enterococci tested (61 E. faecium, 77 E. faecalis, 9 E. gallinarum, and 12 E. casseliflavus isolates). Interestingly, the preliminary sequence comparison of the amplicons for four enterococcal species demonstrated that there were some sequence variations which may be used to generate species-specific internal probes. In conclusion, this rapid PCR

  12. PCR detection of bovine herpesviruses from nonbovine ruminants in Hungary.

    PubMed

    Kálmán, Dóra; Egyed, László

    2005-07-01

    Polymerase chain reaction (PCR) was used to test six different nonbovine ruminant species for five bovine herpesviruses including infectious bovine rhinotracheitis virus (BoHV-1), bovine herpes mammillitis virus (BoHV-2), Movar-type herpesvirus (BoHV-4), bovine herpesvirus type 5 (BoHV-5), and alcelaphine herpesvirus 1 (AlHV-1). Species tested included 56 roe deer (Capreolus capreolus), 66 red deer (Cervus elaphus), 20 fallow deer (Dama dama), 16 mouflon (Ovis musimon), 34 domestic sheep, and 44 domestic goats, which were sampled in Hungary in 2003. Tracheal and popliteal lymph nodes collected from these animals were tested for the presence of the five bovine herpesviruses using three nested (two of which were duplex) PCR assays. Three bovine herpesviruses (BoHV-1, -2, and -4) were detected, whereas no evidence of AlHV-1 or BoHV-5 was observed. Prevalence of BoHV-1 ranged from 12% to 47%, and PCR-positive results were observed in all species tested. BoHV-2 was detected from roe deer, red deer, fallow deer, mouflon, and domestic sheep, and the prevalence in these species ranged from 3% to 50%. BoHV-4 was detected in all species, with prevalence ranging from 12% to 69%. Sequenced PCR products were 99-100% identical to bovine herpesviral sequences deposited in the GenBank. PMID:16244057

  13. FTA card utility for PCR detection of Mycobacterium leprae.

    PubMed

    Aye, Khin Saw; Matsuoka, Masanori; Kai, Masanori; Kyaw, Kyaw; Win, Aye Aye; Shwe, Mu Mu; Thein, Min; Htoo, Maung Maung; Htoon, Myo Thet

    2011-01-01

    The suitability of the FTA® elute card for the collection of slit skin smear (SSS) samples for PCR detection of Mycobacterium leprae was evaluated. A total of 192 SSS leprosy samples, of bacillary index (BI) 1 to 5, were collected from patients attending two skin clinics in Myanmar and preserved using both FTA® elute cards and 70% ethanol tubes. To compare the efficacy of PCR detection of DNA from each BI class, PCR was performed to amplify an M. leprae-specific repetitive element. Of the 192 samples, 116 FTA® elute card and 112 70% ethanol samples were PCR positive for M. leprae DNA. When correlated with BI, area under the curve (AUC) values of the respective receiver-operating characteristic curves were similar for the FTA® elute card and ethanol collection methods (AUC=0.6). Taken together, our results indicate that the FTA® elute card, which enables the collection, transport, and archiving of clinical samples, is an attractive alternative to ethanol preservation for the detection of M. leprae DNA. PMID:21617312

  14. PCR and real-time PCR assays to detect fungi of Alternaria alternata species.

    PubMed

    Kordalewska, Milena; Brillowska-Dąbrowska, Anna; Jagielski, Tomasz; Dworecka-Kaszak, Bożena

    2015-01-01

    Fungi of the Alternaria genus are mostly associated with allergic diseases. However, with a growing number of immunocompromised patients, these fungi, with A. alternata being the most prevalent one, are increasingly recognized as etiological agents of infections (phaeohyphomycoses) in humans. Nowadays, identification of Alternaria spp. requires their pure culture and is solely based on morphological criteria. Clinically, Alternaria infections may be indistinguishable from other fungal diseases. Therefore, a diagnostic result is often delayed or even not achieved at all. In this paper we present easy to perform and interpret PCR and real-time PCR assays enabling detection of A. alternata species. On the basis of alignment of β-tubulin gene sequences, A. alternata-specific primers were designed. DNA from fungal isolates, extracted in a two-step procedure, were used in PCR and real-time PCR assays followed by electrophoresis or melting temperature analysis, respectively. The assays specificity was confirmed, since positive results were obtained for all A. alternata isolates, and no positive results were obtained neither for other molds, dermatophytes, yeast-like fungi, nor human DNA. The assays developed here enable fast and unambiguous identification of A. alternata pathogens. PMID:26610309

  15. Novel real-time PCR detection assay for Brucella suis

    PubMed Central

    Hänsel, C.; Mertens, K.; Elschner, M. C.; Melzer, F.

    2015-01-01

    Introduction Brucella suis is the causative agent of brucellosis in suidae and is differentiated into five biovars (bv). Biovars 1 and 3 possess zoonotic potential and can infect humans, whereas biovar 2 represents the main source of brucellosis in feral and domestic pigs in Europe. Both aspects, the zoonotic threat and the economic loss, emphasize the necessity to monitor feral and domestic pig populations. Available serological or PCR based methods lack sensitivity and specificity. Results Here a bioinformatics approach was used to identify a B. suis specific 17 bp repeat on chromosome II (BS1330_II0657 locus). This repeat is common for B. suis bv 1 to 4 and was used to develop a TaqMan probe assay. The average PCR efficiency was determined as 95% and the limit of detection as 12,5 fg/µl of DNA, equally to 3.7 bacterial genomes. This assay has the highest sensitivity of all previously described B. suis specific PCR assays, making it possible to detect 3-4 bacterial genomes per 1 µl of sample. The assay was tested 100% specific for B. suis and negative for other Brucella spp. and closely related non-Brucella species. Conclusions This novel qPCR assay could become a rapid, inexpensive and reliable screening method for large sample pools of B. suis 1 to 4. This method will be applicable for field samples after validation. PMID:26392898

  16. Detection and counting of Nitrobacter populations in soil by PCR.

    PubMed Central

    Degrange, V; Bardin, R

    1995-01-01

    Although the biological conversion of nitrite to nitrate is a well-known process, studies of Nitrobacter populations are hindered by their physiological characteristics. This report describes a new method for detecting and counting Nitrobacter populations in situ with the PCR. Two primers from the 16S rRNA gene were used to generate a 397-bp fragment by amplification of Nitrobacter species DNA. No signal was detected from their phylogenetic neighbors or the common soil bacteria tested. Extraction and purification steps were optimized for minimal loss and maximal purity of soil DNA. The detection threshold and accuracy of the molecular method were determined from soil inoculated with 10, 10(2), or 10(3) Nitrobacter hamburgensis cells per g of soil. Counts were also done by the most-probable-number (MPN)-Griess and fluorescent antibody methods. PCR had a lower detection threshold (10(2) Nitrobacter cells per g of soil) than did the MPN-Griess or fluorescent antibody method. When PCR amplification was coupled with the MPN method, the counting rate reached 65 to 72% of inoculated Nitrobacter cells. Tested on nonsterile soil, this rapid procedure was proved efficient. PMID:7793930

  17. [Detection of fish DNA in ruminant feed by PCR amplification].

    PubMed

    Nomura, Tetsuya; Kusama, Toyoko; Kadowaki, Koh-ichi

    2006-10-01

    The Japanese Government has prohibited the use of seafood protein, as well as mammalian protein, in ruminant feed. There is an official method to detect meat and bone meal, but no method is yet available to detect fishmeal in ruminant feed. We tried to develop a suitable method to detect fishmeal in ruminant feed, similar to the official method "PCR detection of animal-derived DNA in feed". Our previously reported primers (fishcon5 and fishcon3-1) showed low sensitivity, so we designed new primers based on a DNA sequence from yellowfin tuna mitchondrial DNA. Among the primers, FM5 and FM3 specifically detected fish DNA (sardine, yellowfin tuna, skipjack tuna, chub mackerel, Pacific saury, salmon, rainbow trout, Japanese anchovy, codfish and Japanese horse mackerel) from fish meat, and did not amplify DNA from animals and plants. The sensitivity for detection of the presence of fishmeal in ruminant feed was 0.01-0.001%. PMID:17128872

  18. [PCR-based detection of pathogens in clinical rheumatology].

    PubMed

    Ehrenstein, B; Reischl, U

    2016-05-01

    In the differential diagnostics of autoimmune-mediated rheumatic diseases, rheumatologists often have to consider infections (e. g. Lyme arthritis) or reactive diseases (e. g. reactive arthritis after urogenital bacterial infections). Furthermore, infections with an atypical presentation or caused by atypical pathogens (opportunistic infections) can complicate the immunosuppressive therapy of autoimmune diseases. For this purpose not only conventional microbiological culture methods but also PCR-based methods are increasingly being applied for the direct detection of pathogens in clinical specimens. The aim of this overview is to present commonly used PCR methods in the clinical practice of rheumatology and to describe their benefits and limitations compared to culture-based detection methods. PMID:26892924

  19. Multiplex PCR for detection of acquired carbapenemase genes.

    PubMed

    Poirel, Laurent; Walsh, Timothy R; Cuvillier, Vincent; Nordmann, Patrice

    2011-05-01

    A rapid and reliable PCR-based technique was developed for detection of genes encoding carbapenemases belonging to different classes. Primers were designed to amplify the following 11 genes: bla(IMP), bla(VIM), bla(NDM), bla(SPM), bla(AIM), bla(DIM), bla(GIM), bla(SIM)bla(KPC), bla(BIC), and bla(OXA-48). Three different multiplex reaction mixtures were defined and evaluated for the detection of all these 11 genes. Using optimized conditions, each reaction mixture allowed to identify the respective genes, with PCR giving distinct amplicon sizes corresponding to the different genes for each mixture. We reported here a rapid and reliable technique for screening all clinically relevant carbapenemase genes. PMID:21398074

  20. Stabilized, Freeze-Dried PCR Mix for Detection of Mycobacteria

    PubMed Central

    Klatser, Paul R.; Kuijper, Sjoukje; van Ingen, Cor W.; Kolk, Arend H. J.

    1998-01-01

    We report here the development of a freeze-drying procedure allowing stabilization at ambient temperature of preoptimized, premixed, and predispensed PCR mixes aimed at the detection of mycobacteria in clinical materials. The freeze-dried mixes retained activity at 4°C and at 20°C for 1 year and for 3 months at 37°C, as judged by their performance with 50 and 500 fg of purified Mycobacterium bovis BCG target DNA. PMID:9620427

  1. PCR detection and characterization of type-2 porcine circovirus.

    PubMed Central

    Hamel, A L; Lin, L L; Sachvie, C; Grudeski, E; Nayar, G P

    2000-01-01

    A polymerase chain reaction (PCR) assay was developed for detecting porcine circovirus (PCV). The assay readily detected type-2 PCV (PCV-2) and type-1 PCV (PCV-1). The PCR primers were designed based on DNA sequences conserved in all reported PCV genomes. Type 1 PCV and type 2 PCV both produced 438 bp amplification products, which were easily identified and differentiated from one another by restriction fragment length polymorphism (RFLP) analysis. Porcine circovirus was detected in 55% (931/1693) of randomly tested pigs with various clinical signs and lesions, most of which were difficult to differentiate from those associated with porcine reproductive and respiratory syndrome (PRRS). The PCR products from all positive clinical samples were identified by RFLP to be only PCV-2; DNA tested by PCR was extracted directly from one or more of lung, mesenteric or mediastinal lymph nodes, and tonsil. Type 2 PCV was also detected in 6% (2/34) of DNA extracted directly from semen of randomly chosen healthy boars. Positive PCR reactions from 554 diseased pigs were characterized by RFLP and categorized into 5 different profiles (A-E), of which 82.8% were PCV-2A (456/554), 3.0% were PCV-2B (17/554), 9.9% were PCV-2C (55/554), 1.1% were PCV-2D (6/554), and 3.2% were PCV-2E (18/554). The complete genomic nucleotide sequences of PCV-2A, B, C, D, and E were determined and found to have at least 95% homology compared with one another and with all other PCV-2 found in the GenBank database. All PCV-2 had less than 76% homology with PCV-1. This PCR assay will hopefully be useful to veterinary diagnostic laboratories for routine testing and surveillance of infection with PCV-2. The RFLP profiling system might be useful for preliminary characterization and identification of PCV isolates and might also benefit studies on the molecular epidemiology of PCV. Images Figure 1. PMID:10680656

  2. Detection of major diarrheagenic bacterial pathogens by multiplex PCR panels.

    PubMed

    Sjöling, Åsa; Sadeghipoorjahromi, Leila; Novak, Daniel; Tobias, Joshua

    2015-03-01

    Diarrheal diseases remain a major threat to the youngest population in low- and middle-income countries. The main bacterial pathogens causing diarrhea are diarrheagenic Escherichia coli (DEC) that consists of enteroaggregative (EAEC), enteropathogenic (EPEC), enterotoxigenic (ETEC), enterohemorrhagic EHEC and enteroinvasive E. coli (EIEC), Salmonella, Shigella spp. (S. dysenteria, S. sonnei, S. flexneri) Campylobacter (C. coli, C. jejuni), Vibrio (V. vulnificus, V. parahaemolyticusm, V. cholerae), Yersinia enterocolitica and Aeromonas hydrophila. The aim of this study was to set up rapid multiplex PCR (mPCR) panels to identify these diarrheagenic pathogens based on their specific virulence genes. Primers against specific target genes were combined into three mPCR panels: one for diarrheal E. coli, one for pathogens causing mainly bloody diarrhea, and the third for the remaining pathogens. The panels were tested against a set of stool samples from Swedish children with diarrhea and controls and the analysis identified bacterial pathogens in 14/54 (26%) of the samples. These results show that our three developed mPCR panels can detect main bacterial diarrheagenic pathogens in clinical samples. PMID:25542594

  3. [Detection of Staphylococcus aureus toxins using immuno-PCR].

    PubMed

    Maerle, A V; Riazantsev, D Iu; Dmitrenko, O A; Petrova, E Ia; Komaleva, R L; Sergeev, I V; Trofimov, D Iu; Zavriev, S K

    2014-01-01

    A highly sensitive test system, based on the method of immuno-PCR, was developed for the detection of two staphylococcal toxins: enterotoxin A (SEA) and toxic shock syndrome toxin (TSST). A key element of the developed systems was obtaining supramolecular complexes of bisbiotinylated oligodeoxynucleotides and streptavidin, which were used as DNA tags. Specificity studies showed no cross-reactivity when determining SEA and TSST. The sensitivity of detection of these toxins in the culture supernatants S. aureus was not less than 10 pg/mL. PMID:25895352

  4. TqPCR: A Touchdown qPCR Assay with Significantly Improved Detection Sensitivity and Amplification Efficiency of SYBR Green qPCR

    PubMed Central

    Zhang, Qian; Wang, Jing; Deng, Fang; Yan, Zhengjian; Xia, Yinglin; Wang, Zhongliang; Ye, Jixing; Deng, Youlin; Zhang, Zhonglin; Qiao, Min; Li, Ruifang; Denduluri, Sahitya K.; Wei, Qiang; Zhao, Lianggong; Lu, Shun; Wang, Xin; Tang, Shengli; Liu, Hao; Luu, Hue H.; Haydon, Rex C.; He, Tong-Chuan; Jiang, Li

    2015-01-01

    The advent of fluorescence-based quantitative real-time PCR (qPCR) has revolutionized the quantification of gene expression analysis in many fields, including life sciences, agriculture, forensic science, molecular diagnostics, and medicine. While SYBR Green-based qPCR is the most commonly-used platform due to its inexpensive nature and robust chemistry, quantifying the expression of genes with low abundance or RNA samples extracted from highly restricted or limited sources can be challenging because the detection sensitivity of SYBR Green-based qPCR is limited. Here, we develop a novel and effective touchdown qPCR (TqPCR) protocol by incorporating a 4-cycle touchdown stage prior to the quantification amplification stage. Using the same cDNA templates, we find that TqPCR can reduce the average Cq values for Gapdh, Rps13, and Hprt1 reference genes by 4.45, 5.47, and 4.94 cycles, respectively, when compared with conventional qPCR; the overall average Cq value reduction for the three reference genes together is 4.95. We further find that TqPCR can improve PCR amplification efficiency and thus increase detection sensitivity. When the quantification of Wnt3A-induced target gene expression in mesenchymal stem cells is analyzed, we find that, while both conventional qPCR and TqPCR can detect the up-regulation of the relatively abundant target Axin2, only TqPCR can detect the up-regulation of the lowly-expressed targets Oct4 and Gbx2. Finally, we demonstrate that the MRQ2 and MRQ3 primer pairs derived from mouse reference gene Tbp can be used to validate the RNA/cDNA integrity of qPCR samples. Taken together, our results strongly suggest that TqPCR may increase detection sensitivity and PCR amplification efficiency. Overall, TqPCR should be advantageous over conventional qPCR in expression quantification, especially when the transcripts of interest are lowly expressed, and/or the availability of total RNA is highly restricted or limited. PMID:26172450

  5. Detection of beet yellows virus by RT-PCR and immunocapture RT-PCR in Tetragonia expansa and Beta vulgaris.

    PubMed

    Kundu, K; Rysánek, P

    2004-01-01

    Two sensitive methods, RT-PCR with phenol-extracted RNA or Triton X-100-released RNA and immunocapture RT-PCR (IR-RT-PCR) were used for the detection of Beet yellows virus (BYV) in young and old leaves of Tetragonia expansa and sugar beet (Beta vulgaris) and in sugar beet roots. Four oligonucleotide primer pairs proved suitable for the detection of BYV. The release of BYV RNA with Triton X-100 was shown to be a very effective and easy as compared to isolation of total RNA by phenol extraction with the same or higher sensitivity of subsequent PCR. Using the Triton X-100 release of RNA and IC-RT-PCR the sensitivity of detection was so high that pg amounts of BYV RNA occurring in dilutions up to 10(-6) of saps from young Tetragonia and sugar beet leaves could be detected. PMID:15595212

  6. Real-time PCR detection of ruminant DNA.

    PubMed

    Mendoza-Romero, Luis; Verkaar, Edward L C; Savelkoul, Paul H; Catsburg, Arnold; Aarts, Henk J M; Buntjer, Jaap B; Lenstra, Johannes A

    2004-03-01

    To control the spread of bovine spongiform encephalopathy, several DNA methods have been described for the detection of the species origin of meat and bone meal. Most of these methods are based on the amplification of a mitochondrial DNA segment. We have developed a semiquantitative method based on real-time PCR for detection of ruminant DNA, targeting an 88-bp segment of the ruminant short interspersed nuclear element Bov-A2. This method is specific for ruminants and is able to detect as little as 10 fg of bovine DNA. Autoclaving decreased the amount of detectable DNA, but positive signals were observed in feeding stuff containing 10% bovine material if this had not been rendered in accordance with the regulations, i.e., heated at 134 degrees C for 3 instead of 20 min. PMID:15035372

  7. Nested PCR for detection of HSV-1 in oral mucosa

    PubMed Central

    Jalouli, Miranda-Masoumeh; Jalouli, Jamshid; Hasséus, Bengt; Öhman, Jenny; Hirsch, Jan-Michaél

    2015-01-01

    Background It has been estimated that 15%-20% of human tumours are driven by infection and inflammation, and viral infections play an important role in malignant transformation. The evidence that herpes simplex virus type 1 (HSV-1) could be involved in the aetiology of oral cancer varies from weak to persuasive. This study aimed to investigate by nested PCR (NPCR) the prevalence of HSV-1 in samples from normal oral mucosa, oral leukoplakia, and oral squamous cell carcinoma (OSCC). Material and Methods We investigated the prevalence of HSV-1 in biopsies obtained from 26 fresh, normal oral mucosa from healthy volunteers as well as 53 oral leukoplakia and 27 OSCC paraffin-embedded samples. DNA was extracted from the specimens and investigated for the presence of HSV-1 by nested polymerase chain reaction (NPCR) and DNA sequencing. Results HSV-1 was detected in 14 (54%) of the healthy samples, in 19 (36%) of the oral leukoplakia samples, and in 14 (52%) of the OSCC samples. The differences were not statistically significant. Conclusions We observed a high incidence of HSV-1 in healthy oral mucosa, oral leukoplakia, and OSCC tissues. Thus, no connection between OSCC development and presence of HSV-1 was detected. Key words:HSV-1, nested PCR, PCR. PMID:26449432

  8. Usefulness of PCR method for detection of Leishmania in Poland.

    PubMed

    Myjak, Przemysław; Szulta, Joanna; de Almeida, Marcos E; da Silva, Alexandre J; Steurer, Francis; Lass, Anna; Pietkiewicz, Halina; Nahorski, Wacław L; Goljan, Jolanta; Knap, Józef; Szostakowska, Beata

    2009-01-01

    Leishmania parasites are the etiological agents of leishmaniosis, with severe course and often fatal prognosis, and the global number of cases has increased in recent decades. The gold standards for the diagnosis of leishmaniosis are microscopic examinations and culture in vitro of the different clinical specimens. The sensitivity of these methods is insufficient. Recent development in specific and sensitive molecular methods (PCR) allows for detection as well as identification of the parasite species (subspecies). The aim of the study was to estimate the usefulness of molecular methods (PCR) for detection of Leishmania species and consequently for the implementation of such methods in routine diagnostics of leishmaniosis in Polish patients returning from endemic areas of the disease. In our investigations we used 54 known Leishmania positive DNA templates (from culture and clinical specimens) received from the CDC (Atlanta, GA, USA). Moreover, 25 samples of bone marrow, blood or other tissues obtained from 18 Polish individuals suspected of leishmaniosis were also examined. In PCR we used two pairs of primers specific to the conserved region of Leishmania kinetoplast DNA (kDNA) minicircle (13A/13B and F/R). Using these primers we obtained amplicons in all DNA templates from the CDC and in three Polish patients suspected for Leishmania infection. In one sample from among these cases we also obtained positive results with DNA isolated from a blood specimen which was previously negative in microscopic examinations. PMID:19899614

  9. Immunoliposome-PCR: a generic ultrasensitive quantitative antigen detection system

    PubMed Central

    2012-01-01

    Background The accurate quantification of antigens at low concentrations over a wide dynamic range is needed for identifying biomarkers associated with disease and detecting protein interactions in high-throughput microarrays used in proteomics. Here we report the development of an ultrasensitive quantitative assay format called immunoliposome polymerase chain reaction (ILPCR) that fulfills these requirements. This method uses a liposome, with reporter DNA encapsulated inside and biotin-labeled polyethylene glycol (PEG) phospholipid conjugates incorporated into the outer surface of the liposome, as a detection reagent. The antigenic target is immobilized in the well of a microplate by a capture antibody and the liposome detection reagent is then coupled to a biotin-labeled second antibody through a NeutrAvidin bridge. The liposome is ruptured to release the reporter DNA, which serves as a surrogate to quantify the protein target using real-time PCR. Results A liposome detection reagent was prepared, which consisted of a population of liposomes ~120 nm in diameter with each liposome possessing ~800 accessible biotin receptors and ~220 encapsulated reporters. This liposome detection reagent was used in an assay to quantify the concentration of carcinoembryonic antigen (CEA) in human serum. This ILPCR assay exhibited a linear dose–response curve from 10-10 M to 10-16 M CEA. Within this range the assay coefficient of variance was <6 % for repeatability and <2 % for reproducibility. The assay detection limit was 13 fg/mL, which is 1,500-times more sensitive than current clinical assays for CEA. An ILPCR assay to quantify HIV-1 p24 core protein in buffer was also developed. Conclusions The ILPCR assay has several advantages over other immuno-PCR methods. The reporter DNA and biotin-labeled PEG phospholipids spontaneously incorporate into the liposomes as they form, simplifying preparation of the detection reagent. Encapsulation of the reporter inside the

  10. Simultaneous detection of bee viruses by multiplex PCR.

    PubMed

    Sguazza, Guillermo Hernán; Reynaldi, Francisco José; Galosi, Cecilia Mónica; Pecoraro, Marcelo Ricardo

    2013-12-01

    Honey bee mortality is a serious problem that beekeepers in Argentina have had to face during the last 3 years. It is known that the consequence of the complex interactions between environmental and beekeeping parameters added to the effect of different disease agents such as viruses, bacteria, fungi and parasitic mites may result in a sudden collapse of the colony. In addition, multiple viral infections are detected frequently concomitantly in bee colonies. The aim of this study was to establish a multiplex polymerase chain reaction method for rapid and simultaneous detection of the most prevalent bee viruses. This multiplex PCR assay will provide specific, rapid and reliable results and allow for the cost effective detection of a particular virus as well as multiple virus infections in a single reaction tube. This method could be a helpful tool in the surveillance of the most frequently found bee viruses and to study the dynamics and the interactions of the virus populations within colonies. PMID:23948157

  11. Fast detection of deletion breakpoints using quantitative PCR.

    PubMed

    Abildinova, Gulshara; Abdrakhmanova, Zhanara; Tuchinsky, Helena; Nesher, Elimelech; Pinhasov, Albert; Raskin, Leon

    2016-06-16

    The routine detection of large and medium copy number variants (CNVs) is well established. Hemizygotic deletions or duplications in the large Duchenne muscular dystrophy DMD gene responsible for Duchenne and Becker muscular dystrophies are routinely identified using multiple ligation probe amplification and array-based comparative genomic hybridization. These methods only map deleted or duplicated exons, without providing the exact location of breakpoints. Commonly used methods for the detection of CNV breakpoints include long-range PCR and primer walking, their success being limited by the deletion size, GC content and presence of DNA repeats. Here, we present a strategy for detecting the breakpoints of medium and large CNVs regardless of their size. The hemizygous deletion of exons 45-50 in the DMD gene and the large autosomal heterozygous PARK2 deletion were used to demonstrate the workflow that relies on real-time quantitative PCR to narrow down the deletion region and Sanger sequencing for breakpoint confirmation. The strategy is fast, reliable and cost-efficient, making it amenable to widespread use in genetic laboratories. PMID:27333265

  12. Fast detection of deletion breakpoints using quantitative PCR

    PubMed Central

    Abildinova, Gulshara; Abdrakhmanova, Zhanara; Tuchinsky, Helena; Nesher, Elimelech; Pinhasov, Albert; Raskin, Leon

    2016-01-01

    Abstract The routine detection of large and medium copy number variants (CNVs) is well established. Hemizygotic deletions or duplications in the large Duchenne muscular dystrophy DMD gene responsible for Duchenne and Becker muscular dystrophies are routinely identified using multiple ligation probe amplification and array-based comparative genomic hybridization. These methods only map deleted or duplicated exons, without providing the exact location of breakpoints. Commonly used methods for the detection of CNV breakpoints include long-range PCR and primer walking, their success being limited by the deletion size, GC content and presence of DNA repeats. Here, we present a strategy for detecting the breakpoints of medium and large CNVs regardless of their size. The hemizygous deletion of exons 45-50 in the DMD gene and the large autosomal heterozygous PARK2 deletion were used to demonstrate the workflow that relies on real-time quantitative PCR to narrow down the deletion region and Sanger sequencing for breakpoint confirmation. The strategy is fast, reliable and cost-efficient, making it amenable to widespread use in genetic laboratories. PMID:27560363

  13. Fast detection of deletion breakpoints using quantitative PCR.

    PubMed

    Abildinova, Gulshara; Abdrakhmanova, Zhanara; Tuchinsky, Helena; Nesher, Elimelech; Pinhasov, Albert; Raskin, Leon

    2016-01-01

    The routine detection of large and medium copy number variants (CNVs) is well established. Hemizygotic deletions or duplications in the large Duchenne muscular dystrophy DMD gene responsible for Duchenne and Becker muscular dystrophies are routinely identified using multiple ligation probe amplification and array-based comparative genomic hybridization. These methods only map deleted or duplicated exons, without providing the exact location of breakpoints. Commonly used methods for the detection of CNV breakpoints include long-range PCR and primer walking, their success being limited by the deletion size, GC content and presence of DNA repeats. Here, we present a strategy for detecting the breakpoints of medium and large CNVs regardless of their size. The hemizygous deletion of exons 45-50 in the DMD gene and the large autosomal heterozygous PARK2 deletion were used to demonstrate the workflow that relies on real-time quantitative PCR to narrow down the deletion region and Sanger sequencing for breakpoint confirmation. The strategy is fast, reliable and cost-efficient, making it amenable to widespread use in genetic laboratories. PMID:27560363

  14. Rapid detection of Serpulina hyodysenteriae in diagnostic specimens by PCR.

    PubMed Central

    Elder, R O; Duhamel, G E; Schafer, R W; Mathiesen, M R; Ramanathan, M

    1994-01-01

    A PCR assay for the detection of Serpulina hyodysenteriae in diagnostic specimens was developed on the basis of sequence analysis of a recombinant clone designated pRED3C6. Clone pRED3C6, which contained a 2.3-kb DNA fragment unique to S. hyodysenteriae, was identified by screening a plasmid library of S. hyodysenteriae isolate B204 genomic DNA in Escherichia coli by colony immunoblot with the mouse monoclonal antibody 10G6/G10, which was produced against cell-free supernatant antigens from the same isolate. Southern blot analysis of HindIII-digested genomic DNA of S. hyodysenteriae serotypes 1 through 7 and of four weakly beta-hemolytic intestinal spirochetes, including Serpulina innocens, with the 2.3-kb DNA fragment of pRED3C6 indicated that the cloned sequence was present exclusively in the seven serotypes of S. hyodysenteriae. An oligonucleotide primer pair for PCR amplification of a 1.55-kb fragment and an internal oligonucleotide probe were designed and synthesized on the basis of sequence analysis of the 2.3-kb DNA fragment of pRED3C6. Purified genomic DNAs from reference isolates of S. hyodysenteriae serotypes 1 through 9, S. innocens, weakly beta-hemolytic intestinal spirochetes belonging to genotypic groups distinct from those of reference Serpulina spp., other cultivable reference isolates of the order Spirochaetales, and enteric bacteria including Escherichia coli, Salmonella spp., Campylobacter spp., and Bacteroides vulgatus were amplified with the oligonucleotide primer pair in a hot-start PCR. The 1.55-kb products were obtained only in the presence of genomic DNA from each of the nine serotypes of S. hyodysenteriae. The specificity of the 1.55-kb products for S. hyodysenteriae was confirmed on the basis of production of a restriction endonuclease pattern of the PCR products identical to the predicted restriction map analysis of pRED3C6 and positive hybridization signal with the S. hyodysenteriae-specific internal oligonucleotide probe. By using

  15. Multiplex PCR method for detection of three Aeromonas enterotoxin genes.

    PubMed

    Kingombe, Cesar I Bin; D'Aoust, Jean-Yves; Huys, Geert; Hofmann, Lisa; Rao, Mary; Kwan, Judy

    2010-01-01

    A novel multiplex PCR method using three sets of specific primers was developed for the detection of the cytotoxic (act), heat-labile (alt), and heat-stable (ast) enterotoxin genes in Aeromonas spp. This assay was used to characterize 35 reference strains as well as 537 food-borne isolates. A total of seven gene pattern combinations were encountered, including act, alt, act/alt, act/alt/ast, act/alt/148-bp amplicon, alt/ast, and alt/148-bp amplicon. The alt gene was detected with 34 reference strains (97%) and occurred singly in 14% of these strains. The frequency of occurrence of the act/alt, act/alt/ast, and alt/ast gene patterns in reference strains was 14 (40%), 2 (6%), and 2 (6%), respectively. An unpredicted amplicon was detected in 11 reference strains (31%). Characterization of this amplicon showed that its size was 148 bp, as generated by the AHLF and AHLR primers, and that it uniquely aligned with the Aeromonas salmonicida A449 genome sequence (GenBank accession number CP000644). This amplicon was named Aeromonas salmonicida subsp. salmonicida hypothetical protein amplicon (AssHPA). In the 537 food-borne isolates, the act and alt genes were most dominant and were detected in 349 (65%) and 452 (84%) isolates, respectively, either alone or in combinations. The act and alt genes occurred singly in 30 (6%) and 128 (24%) of these strains, respectively. The act/alt gene pattern occurred in 315 isolates (59%), whereas the ast gene was always linked to strains exhibiting the act/alt/ast and alt/ast gene combinations in 4 (0.7%) and 5 (0.9%) isolates, respectively. The uniplex amplification of three enterotoxin genes separately confirms the specificity of the unique selected primers. This multiplex PCR is rapid and simple and can detect the presence of three Aeromonas enterotoxin genes in a single assay. PMID:19933350

  16. Comparison of Conventional PCR, Multiplex PCR, and Loop-Mediated Isothermal Amplification Assays for Rapid Detection of Arcobacter Species

    PubMed Central

    Wang, Xiaoyu; Seo, Dong Joo; Lee, Min Hwa

    2014-01-01

    This study aimed to develop a loop-mediated isothermal amplification (LAMP) method for the rapid detection of Arcobacter species. Specific primers targeting the 23S ribosomal RNA gene were used to detect Arcobacter butzleri, Arcobacter cryaerophilus, and Arcobacter skirrowii. The specificity of the LAMP primer set was assessed using DNA samples from a panel of Arcobacter and Campylobacter species, and the sensitivity was determined using serial dilutions of Arcobacter species cultures. LAMP showed a 10- to 1,000-fold-higher sensitivity than multiplex PCR, with a detection limit of 2 to 20 CFU per reaction in vitro. Whereas multiplex PCR showed cross-reactivity with Campylobacter species, the LAMP method developed in this study was more sensitive and reliable than conventional PCR or multiplex PCR for the detection of Arcobacter species. PMID:24478488

  17. Detection of Bacteroides fragilis Enterotoxin Gene by PCR

    PubMed Central

    Shetab, Razeq; Cohen, Stuart H.; Prindiville, Thomas; Tang, Yajarayma J.; Cantrell, Mary; Rahmani, Darush; Silva, Joseph

    1998-01-01

    Bacteroides fragilis constitutes about 1% of the bacterial flora in intestines of normal humans. Enterotoxigenic strains of B. fragilis have been associated with diarrheal diseases in humans and animals. The enterotoxin produced by these isolates induces fluid changes in ligated intestinal loops and an in vitro cytotoxic response in HT-29 cells. We developed a nested PCR to detect the enterotoxin gene of B. fragilis in stool specimens. After DNA extraction, a 367-bp fragment was amplified with two outer primers. The amplicon from this reaction was subjected to a second round of amplification with a set of internal primers. With these inner primers, a 290-bp DNA fragment was obtained which was confirmed as part of the B. fragilis enterotoxin gene by Southern blotting with a nonradioactive internal probe and a chemiluminescence system. By this approach, B. fragilis enterotoxin gene sequences were detected in eight known enterotoxigenic human isolates and nine enterotoxigenic horse isolates. No amplification products were obtained from DNA extracted from 28 nonenterotoxigenic B. fragilis isolates or B. distasonis, B. thetaiotaomicron, B. uniformis, B. ovatus, Escherichia coli, or Clostridium difficile. The sensitivity of this assay allowed us to detect as little as 1 pg of enterotoxin DNA sequences or 100 to 1,000 cells of enterotoxigenic B. fragilis/g of stool. Enterotoxin production of all isolates was confirmed in vitro in HT-29 cells. A 100% correlation was obtained between enterotoxin detection by cytotoxin assay and the nested PCR assay. This rapid and sensitive assay can be used to identify enterotoxigenic B. fragilis and may be used clinically to determine the role of B. fragilis in diarrheal diseases. PMID:9620408

  18. PCR and Genotyping for HPV in Cervical Cancer Patients

    PubMed Central

    Prakash, Pradyot; Patne, Shashikant C U; Singh, Ashish Kumar; Kumar, Mohan; Mishra, Mukti Nath; Gulati, Anil Kumar

    2016-01-01

    Aims: To devise nested multiplex polymerase chain reaction (NMPCR) protocol for detection of mucosal human papilloma viruses (HPVs) and typing of HPV-16 and -18 in formalin-fixed, paraffin-embedded (FFPE) tissues of carcinoma cervix (CaCx). Settings and Design: Cross-sectional observational study. Materials and Methods: NMPCR was done for simultaneous detection of HPV, targeting 134 bp L1 capsid gene employing GP+/mGP+ primers and typing of genotypes-16 and -18, targeting E6/E7 gene from 34 FFPE tissue blocks of CaCx and cervical intraepithelial neoplasia (CIN). Detection of 142 bp consensus sequence of L1 capsid gene was performed by nested PCR employing MY/GP+ primers. Sequencing of selected PCR amplicons of the later protocol obtained from control cell line DNA and 5 select samples were done for validation of the NMPCR protocol. Statistical Analysis Used: Calculation of percentage from the Microsoft Excel Software. Results: Of 26 FFPE samples of CaCx, 17 (65.3%) samples were found positive for HPV by NMPCR. Amplicons of 142 bp L1 capsid gene employing MY/GP+ primers were observed in 11 (42.3%) samples of CaCx. Nearly 25% samples of CIN were positive for HPV. On sequence analysis, it was observed that the sample typed as HPV-16 by NMPCR was found to be the same on sequencing of amplicons obtained after MY/GP+ nested PCR. Conclusions: This study indicates the usefulness of our NMPCR protocol for detection of mucosal HPVs and typing of HPV-16 and -18 from FFPE tissue samples of CaCx. The NMPCR protocol may be used to detect HPV and type common genotypes-16 and -18 in fresh tissue of cervical biopsy or scrape samples for screening of CaCx. PMID:27621560

  19. Nucleic acid sequence detection using multiplexed oligonucleotide PCR

    DOEpatents

    Nolan, John P.; White, P. Scott

    2006-12-26

    Methods for rapidly detecting single or multiple sequence alleles in a sample nucleic acid are described. Provided are all of the oligonucleotide pairs capable of annealing specifically to a target allele and discriminating among possible sequences thereof, and ligating to each other to form an oligonucleotide complex when a particular sequence feature is present (or, alternatively, absent) in the sample nucleic acid. The design of each oligonucleotide pair permits the subsequent high-level PCR amplification of a specific amplicon when the oligonucleotide complex is formed, but not when the oligonucleotide complex is not formed. The presence or absence of the specific amplicon is used to detect the allele. Detection of the specific amplicon may be achieved using a variety of methods well known in the art, including without limitation, oligonucleotide capture onto DNA chips or microarrays, oligonucleotide capture onto beads or microspheres, electrophoresis, and mass spectrometry. Various labels and address-capture tags may be employed in the amplicon detection step of multiplexed assays, as further described herein.

  20. An improved, PCR-based strategy for the detection of Trypanosoma cruzi in human blood samples.

    PubMed

    Ribeiro-dos-Santos, G; Nishiya, A S; Sabino, E C; Chamone, D F; Saez-Alquézar, A

    1999-10-01

    Attempts were made to improve the PCR-based detection of Trypanosoma cruzi in blood samples, primarily for screening blood donors. Samples were obtained from candidate donors who were reactive in one or two of three serological tests for Chagas disease (and therefore considered 'indeterminate') or in all three tests (3+). Each sample was then examined using three different, PCR-based techniques: 'PCR-I' (in which the target DNA is a nuclear repetitive sequence); 'PCR-II' [amplifying a conserved region of the T. cruzi kinetoplast DNA (kDNA)]; and 'PCR-III' (a new strategy in which the target kDNA is amplified by 'nested' PCR). Among the samples from 3+ individuals, PCR-I, PCR-II and PCR-III amplified two (3.8%) out of 52, four (4.5%) out of 88, and 27 (25.7%) out of 105 samples tested, respectively. Seven, 69 and 70 samples from 'indeterminate' subjects were tested by PCR-I, PCR-II and PCR-III, respectively; there was not a single positive result by PCR-I or PCR-II, but three (4.3%) of the samples tested by PCR-III were positive. In a reconstruction experiment, in conditions in which PCR-I and PCR-II could not detect 10,000 parasites/ml, PCR-III was able to detect one parasite/ml. Although all three PCR-based strategies examined had rather poor sensitivities, PCR-III was far more sensitive than PCR-I or PCR-II. PMID:10715696

  1. Sensitive Simultaneous Detection of Seven Sexually Transmitted Agents in Semen by Multiplex-PCR and of HPV by Single PCR

    PubMed Central

    de Abreu, André Luelsdorf Pimenta; Irie, Mary Mayumi Taguti; Esquiçati, Isis Baroni; Malagutti, Natália; Vasconcellos, Vinícius Rodrigo Bulla; Discacciati, Michele Garcia; Bonini, Marcelo Gialluisi; Maria-Engler, Silvya Stuchi; Consolaro, Marcia Edilaine Lopes

    2014-01-01

    Sexually transmitted diseases (STDs) may impair sperm parameters and functions thereby promoting male infertility. To date limited molecular studies were conducted to evaluate the frequency and type of such infections in semen Thus, we aimed at conceiving and validating a multiplex PCR (M-PCR) assay for the simultaneous detection of the following STD pathogens in semen: Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma genitalium, Trichomonas vaginalis, Herpes virus simplex (HSV) −1 and −2, and Treponema pallidum; We also investigated the potential usefulness of this M-PCR assay in screening programs for semen pathogens. In addition, we aimed: to detect human Papillomavirus (HPV) and genotypes by single PCR (sPCR) in the same semen samples; to determine the prevalence of the seven STDs, HPV and co-infections; to assess the possibility that these infections affect semen parameters and thus fertility. The overall validation parameters of M-PCR were extremely high including agreement (99.2%), sensitivity (100.00%), specificity (99.70%), positive (96.40%) and negative predictive values (100.00%) and accuracy (99.80%). The prevalence of STDs was very high (55.3%). Furthermore, associations were observed between STDs and changes in semen parameters, highlighting the importance of STD detection in semen. Thus, this M-PCR assay has great potential for application in semen screening programs for pathogens in infertility and STD clinics and in sperm banks. PMID:24921247

  2. 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

    2015-05-01

    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. PMID:25850372

  3. PCR detection of groundwater bacteria associated with colloidal transport

    SciTech Connect

    Cruz-Perez, P.; Stetzenbach, L.D.; Alvarez, A.J.

    1996-02-29

    Colloidal transport may increase the amount of contaminant material than that which could be transported by water flow alone. The role of colloids in groundwater contaminant transport is complicated and may involve many different processes, including sorption of elements onto colloidal particles, coagulation/dissolution, adsorption onto solid surfaces, filtration, and migration. Bacteria are known to concentrate minerals and influence the transport of compounds in aqueous environments and may also serve as organic colloids, thereby influencing subsurface transport of radionuclides and other contaminants. The initial phase of the project consisted of assembling a list of bacteria capable of sequestering or facilitating mineral transport. The development and optimization of the PCR amplification assay for the detection of the organisms of interest, and the examination of regional groundwaters for those organisms, are presented for subsequent research.

  4. Detection of Legionella Contamination in Tabriz Hospitals by PCR Assay

    PubMed Central

    Ghotaslou, Reza; Yeganeh Sefidan, Fatemeh; Akhi, Mohammad Taghi; Soroush, Mohammad Hussein; Hejazi, Mohammad Saeid

    2013-01-01

    Purpose: The present study was designed to evaluate the occurrence of Legionella contamination in the tap water of Tabriz hospitals, Azerbaijan, Iran. Methods: One hundred and forty water samples from diverse water supply systems of 17 hospitals were collected and analyzed for the presence of Legionella spp. by PCR assay. Results: In this study, 10 of 140 (7.1%) samples were positive for Legionella which L. pneumophila was detected in 4 (2.85%) water samples. Conclusion: In conclusion, hospital potable systems are the primary reservoirs for Legionnaires’ disease. This study concludes that Legionella spp. are present in aquatic hospitals environment of Tabriz. Due to the serious risk of infections, it is better to make efforts to eliminate Legionella spp. in water supplies. PMID:24312825

  5. Detection of DNA double-strand breaks and chromosome translocations using ligation-mediated PCR and inverse PCR.

    PubMed

    Villalobos, Michael J; Betti, Christopher J; Vaughan, Andrew T M

    2005-01-01

    Current techniques for examining the global creation and repair of DNA double-strand breaks are restricted in their sensitivity, and such techniques mask any site-dependent variations in breakage and repair rate or fidelity. We present here a system for analyzing the fate of documented DNA breaks, using the MLL gene as an example, through application of ligation-mediated PCR. Here, a simple asymmetric double-stranded DNA adapter molecule is ligated to experimentally induced DNA breaks and subjected to seminested PCR using adapter and gene-specific primers. The rate of appearance and loss of specific PCR products allows detection of both the break and its repair. Using the additional technique of inverse PCR, the presence of misrepaired products (translocations) can be detected at the same site, providing information on the fidelity of the ligation reaction in intact cells. Such techniques may be adapted for the analysis of DNA breaks introduced into any identifiable genomic location. PMID:15502230

  6. Comparing Rapid and Specific Detection of Brucella in Clinical Samples by PCR-ELISA and Multiplex-PCR Method

    PubMed Central

    Mohammad Hasani, Sharareh; Mirnejad, Reza; Amani, Jafar; Vafadar, Mohamad javad

    2016-01-01

    Background: Rapid diagnosis and differentiation of Brucella is of high importance due to the side effects of antibiotics for the treatment of brucellosis. This study aimed to identify and compare PCR-ELISA as a more accurate diagnositc test with other common molecular and serological tests. Methods: In this experimental and sectional study, during March 2014 to Sep 2015, 52 blood samples of suspected patients with clinical symptoms of brucellosis were evaluated in medical centers all over Iran with serum titers higher than 1:80. Using two pairs of specific primers of Brucella abortus, B. melitensis and DIG-dUTP, Fragment IS711 (The common gene fragment in B. melitensis and B. abortus) was amplified. DIG-ELISA was performed using specific probes of these 2 species of Brucella and patterns were subsequently analyzed, then positive responses were compared by detecting gel electrophoresis. Results: PCR-ELISA method detected all 28 samples from 52 positive samples. Its sensitivity was 6.0 pg concentration of genomic DNA of Brucella. In gel electrophoresis method, 22 samples of all positive samples were detected. PCR-ELISA was more efficient than PCR and bacterial culture method at P-value <0.05. Conclusion: PCR-ELISA molecular method is more sensitive than other molecular methods, lack of mutagenic color and also a semi-quantitative ability. This method is more effective and more accurate compared to PCR, serology and culture of bacteria. PCR-ELISA does not have false responses. The limitation of this method is detection of bacteria in the genus compared to Multiplex PCR and Gel Electrophoresis. PMID:27499776

  7. Detection of North American orthopoxviruses by real time-PCR

    PubMed Central

    2011-01-01

    The prevalence of North American orthopoxviruses in nature is unknown and may be more difficult to ascertain due to wide spread use of vaccinia virus recombinant vaccines in the wild. A real time PCR assay was developed to allow for highly sensitive and specific detection of North American orthopoxvirus DNA in animal tissues and bodily fluids. This method is based on the amplification of a 156 bp sequence within a myristylated protein, highly conserved within the North American orthopoxviruses but distinct from orthologous genes present in other orthopoxviruses. The analytical sensitivity was 1.1 fg for Volepox virus DNA, 1.99 fg for Skunkpox virus DNA, and 6.4 fg for Raccoonpox virus DNA with a 95% confidence interval. Our assay did not cross-react with other orthopoxviruses or ten diverse representatives of the Chordopoxvirinae subfamily. This new assay showed more sensitivity than tissue culture tests, and was capable of differentiating North American orthopoxviruses from other members of Orthopoxvirus. Thus, our assay is a promising tool for highly sensitive and specific detection of North American orthopoxviruses in the United States and abroad. PMID:21689420

  8. Detection and quantification of Bacillus cereus group in milk by droplet digital PCR.

    PubMed

    Porcellato, Davide; Narvhus, Judith; Skeie, Siv Borghild

    2016-08-01

    Droplet digital PCR (ddPCR) is one of the newest and most promising methods for the detection and quantification of molecular targets by PCR. Here, we optimized and used a new ddPCR assay for the detection and quantification of the Bacillus cereus group in milk. We also compared the ddPCR to a standard qPCR assay. The new ddPCR assay showed a similar coefficient of determination and a better limit of detection compared to the qPCR assay during quantification of the target molecules in the samples. However, the ddPCR assay has a limitation during quantification of a high number of target molecules. This new assay was then tested for the quantification of the B. cereus group in 90 milk samples obtained over three months from two different dairies and the milk was stored at different temperatures before sampling. The ddPCR assay showed good agreement with the qPCR assay for the quantification of the B. cereus group in milk, and due to its lower detection limit more samples were detected as positive. The new ddPCR assay is a promising method for the quantification of target bacteria in low concentration in milk. PMID:27211508

  9. Detection and quantification of chimerism by droplet digital PCR.

    PubMed

    George, David; Czech, Juliann; John, Bobby; Yu, Min; Jennings, Lawrence J

    2013-01-01

    Accurate quantification of chimerism and microchimerism is proving to be increasingly valuable for hematopoietic cell transplantation as well as non-transplant conditions. However, methods that are available to quantify low-level chimerism lack accuracy. Therefore, we developed and validated a method for quantifying chimerism based on digital PCR technology. We demonstrate accurate quantification that far exceeds what is possible with analog qPCR down to 0.01% with the potential to go even lower. Also, this method is inherently more informative than qPCR. We expect the advantages of digital PCR will make it the preferred method for chimerism analysis. PMID:23974275

  10. Detection of salmonella using a real-time PCR based on molecular beacons

    NASA Astrophysics Data System (ADS)

    Chen, Wilfred; Martinez, Grisselle; Mulchandani, Ashok

    2000-03-01

    Molecular beacons are oligonucleotide probes that become fluorescent upon hybridization. We developed a new approach to detect the presence of Salmonella species using these fluorogenic reporter molecules and demonstrated their ability to discriminate between similar E. coli species in real-time PCR assays. A detection limit of 1 CFU per PCR reaction was obtained. The assays were carried out entirely in sealed PCR tubes, enabling fast and direct detection in a semiautomated format.

  11. Detection and Quantification of Wallemia sebi in Aerosols by Real-Time PCR, Conventional PCR, and Cultivation

    PubMed Central

    Zeng, Qing-Yin; Westermark, Sven-Olof; Rasmuson-Lestander, Åsa; Wang, Xiao-Ru

    2004-01-01

    Wallemia sebi is a deuteromycete fungus commonly found in agricultural environments in many parts of the world and is suspected to be a causative agent of farmer's lung disease. The fungus grows slowly on commonly used culture media and is often obscured by the fast-growing fungi. Thus, its occurrence in different environments has often been underestimated. In this study, we developed two sets of PCR primers specific to W. sebi that can be applied in either conventional PCR or real-time PCR for rapid detection and quantification of the fungus in environmental samples. Both PCR systems proved to be highly specific and sensitive for W. sebi detection even in a high background of other fungal DNAs. These methods were employed to investigate the presence of W. sebi in the aerosols of a farm. The results revealed a high concentration of W. sebi spores, 107 m−3 by real-time PCR and 106 m−3 by cultivation, which indicates the prevalence of W. sebi in farms handling hay and grain and in cow barns. The methods developed in this study could serve as rapid, specific, and sensitive means of detecting W. sebi in aerosol and surface samples and could thus facilitate investigations of its distribution, ecology, clinical diagnosis, and exposure risk assessment. PMID:15574929

  12. Type-A influenza virus detection and quantitation by real-time RT-PCR

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Real-time RT-PCR (RRT-PCR) is a relatively new technology which has been used for AIV detection since the early 2000’s for routine surveillance, during outbreaks and for research. Some of the advantages of RRT-PCR are: quantitative nature, scalability, cost, high sensitivity, high specificity, and ...

  13. Effects of prolonged chlorine exposures upon PCR detection of Helicobacter pylori DNA.

    EPA Science Inventory

    The effect of low doses of free chlorine on the detection by qPCR of Helicobacter pylori (H. pylori) cells by qPCR in tap water was monitored. H. pylori target sequences (within suspended, intact cells at densities of 102 to 103 cells /ml) were rendered undetectable by qPCR an...

  14. DEVELOPMENT OF AN IMPROVED PCR-BASED TECHNIQUE FOR DETECTION OF PHYTOPHTHORA CACTORUM IN STRAWBERRY PLANTS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Specific and rapid plant pathogen detection methods can aid in strawberry disease management decisions. PCR-based diagnostics for Phytophthora cactorum and other strawberry pathogens are hindered by PCR inhibitors and lack of species-specific PCR primers. We developed a DNA extraction and purificati...

  15. Optimized 5-hour multiplex PCR test for the detection of tinea unguium: performance in a routine PCR laboratory.

    PubMed

    Brillowska-Dabrowska, Anna; Nielsen, Sanne Søgaard; Nielsen, Henrik Vedel; Arendrup, Maiken Cavling

    2010-09-01

    We recently reported the development of a 5-hour multiplex PCR test for the detection of tinea unguium and the optimization of this test by the inclusion of an inhibition control. Here we report the performance of this procedure as used in a routine clinical laboratory as compared to conventional microscopy and culture-based techniques performed in a mycology reference laboratory. We found in processing 109 samples that 22 (20.2%) yielded fungi in culture while the suspected etiologic agents were noted microscopically in 15 (13.8%) that were negative in culture. Fungi were detected by PCR in 37 (33.9%) samples, of which only three were positive in culture. Since the majority of PCR positive but culture negative samples were positive in microscopic examinations, the increased sensitivity was not due to contamination. PCR inhibitors were present in 5% of the samples, but this was overcome by re-running the samples with a 50% reduction of sample DNA. In conclusion, the PCR test performance in the routine setting was excellent and provided a markedly reduced time to diagnosis with a higher sensitivity. PMID:20105101

  16. Comparative analysis of human papillomavirus detection by PCR and non-isotopic in situ hybridisation.

    PubMed Central

    Herrington, C S; Anderson, S M; Bauer, H M; Troncone, B; de Angelis, M L; Noell, H; Chimera, J A; Van Eyck, S L; McGee, J O

    1995-01-01

    AIMS--To assess the relative diagnostic performance of the polymerase chain reaction (PCR) and non-isotopic in situ hybridisation (NISH) and to correlate these data with cytopathological assessment. METHODS--Paired analysis of human papillomavirus (HPV) detection was performed by PCR and NISH on exfoliated cervical cells from 122 women attending a routine gynaecological examination. PCR amplification followed by generic and HPV type specific hybridisation was compared with NISH on a parallel cervical smear. RESULTS--Overall, 32 cases were positive by NISH and 61 positive by PCR. Of the 105 cases in which both PCR and NISH were interpretable, 76 (26%) were normal smears, 20 of which were HPV positive by NISH and 37 (49%) by PCR. Of 17 borderline smears, two were NISH positive and 12 PCR positive. Eight of nine smears containing koilocytes were positive by NISH and seven by PCR. Of three dyskaryotic smears, none were NISH and two were PCR positive. The concordance of NISH and PCR in these samples was 57%. To assess sampling error, NISH and PCR were performed on an additional 50 cases using aliquots from the same sample. This increased the concordance between assays to 74%. Filter hybridisation of PCR products with the cocktail of probes used in NISH (under low and high stringency conditions) demonstrated that several cases of NISH positivity could be accounted for by cross-hybridisation to HPV types identified by PCR but not present in the NISH probe cocktail. CONCLUSIONS--Sampling error and potential cross-hybridisation of probe and target should be considered in interpretation of these techniques. PCR is more sensitive because it provides for the amplification of target DNA sequences. In addition, the PCR assay utilised in this study detects a wider range of HPV types than are contained in the cocktails used for NISH. However, PCR assays detect viral DNA present both within cells and in cervical fluid whereas NISH permits morphological localisation. Images PMID

  17. Comparative analysis of human papillomavirus detection by PCR and non-isotopic in situ hybridisation.

    PubMed

    Herrington, C S; Anderson, S M; Bauer, H M; Troncone, B; de Angelis, M L; Noell, H; Chimera, J A; Van Eyck, S L; McGee, J O

    1995-05-01

    AIMS--To assess the relative diagnostic performance of the polymerase chain reaction (PCR) and non-isotopic in situ hybridisation (NISH) and to correlate these data with cytopathological assessment. METHODS--Paired analysis of human papillomavirus (HPV) detection was performed by PCR and NISH on exfoliated cervical cells from 122 women attending a routine gynaecological examination. PCR amplification followed by generic and HPV type specific hybridisation was compared with NISH on a parallel cervical smear. RESULTS--Overall, 32 cases were positive by NISH and 61 positive by PCR. Of the 105 cases in which both PCR and NISH were interpretable, 76 (26%) were normal smears, 20 of which were HPV positive by NISH and 37 (49%) by PCR. Of 17 borderline smears, two were NISH positive and 12 PCR positive. Eight of nine smears containing koilocytes were positive by NISH and seven by PCR. Of three dyskaryotic smears, none were NISH and two were PCR positive. The concordance of NISH and PCR in these samples was 57%. To assess sampling error, NISH and PCR were performed on an additional 50 cases using aliquots from the same sample. This increased the concordance between assays to 74%. Filter hybridisation of PCR products with the cocktail of probes used in NISH (under low and high stringency conditions) demonstrated that several cases of NISH positivity could be accounted for by cross-hybridisation to HPV types identified by PCR but not present in the NISH probe cocktail. CONCLUSIONS--Sampling error and potential cross-hybridisation of probe and target should be considered in interpretation of these techniques. PCR is more sensitive because it provides for the amplification of target DNA sequences. In addition, the PCR assay utilised in this study detects a wider range of HPV types than are contained in the cocktails used for NISH. However, PCR assays detect viral DNA present both within cells and in cervical fluid whereas NISH permits morphological localisation. PMID:7629286

  18. Development of nested PCR assays for detection of bovine respiratory syncytial virus in clinical samples.

    PubMed Central

    Vilcek, S; Elvander, M; Ballagi-Pordány, A; Belák, S

    1994-01-01

    Two nested PCR assays were developed for the detection of bovine respiratory syncytial virus (BRSV). Primers were selected from the gene encoding the F fusion protein (PCR-F) and the gene encoding the G attachment protein (PCR-G). Biotinylated oligonucleotide probes, termed F and G, were selected for the hybridization of the respective PCR products. The sensitivities of the PCR-F and PCR-G assays were similar, both detecting 0.1 tissue culture infective dose of the virus. The PCR-F assay amplified all bovine strains and one human strain (RS32) tested. No cross-reactions were observed with nine heterologous respiratory viruses. PCR-F products of bovine and human RSV strains were discriminated by using endonuclease restriction enzyme ScaI, which specifically cleaved, products of BRSV. Oligonucleotide probe F was also specific for products of BRSV. The PCR-G assay detected all bovine strains and none of the human strains tested. A faint electrophoretic band was also observed with products of Sendai virus. However, probe G did not hybridize with this product, only with products of BRSV. Nasal swabs collected from cattle with no symptoms and cattle in the acute stage of respiratory disease were analyzed for BRSV by the immunofluorescence (IF) method and by the PCR-F and PCR-G assays. The virus was detected by the PCR assays in 31 of 35 (89%) samples tested. Only 23 samples (66%) were positive by the IF method, and these samples were also positive by both the PCR-F and PCR-G assays. The 31 samples detected as positive by PCR originated from cattle presenting clinical signs of acute respiratory disease; the four PCR-negative samples originated from clinically asymptomatic neighboring cattle. All sampled animals subsequently seroconverted and became reactive to BRSV. Thus, the detection of BRSV by PCR correlated with clinical observations and was considerably more sensitive (66 versus 89%) than IF. These results indicate that both nested PCR assays provide rapid and

  19. 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. PMID:26185125

  20. 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.

  1. Comprehensive GMO detection using real-time PCR array: single-laboratory validation.

    PubMed

    Mano, Junichi; Harada, Mioko; Takabatake, Reona; Furui, Satoshi; Kitta, Kazumi; Nakamura, Kosuke; Akiyama, Hiroshi; Teshima, Reiko; Noritake, Hiromichi; Hatano, Shuko; Futo, Satoshi; Minegishi, Yasutaka; Iizuka, Tayoshi

    2012-01-01

    We have developed a real-time PCR array method to comprehensively detect genetically modified (GM) organisms. In the method, genomic DNA extracted from an agricultural product is analyzed using various qualitative real-time PCR assays on a 96-well PCR plate, targeting for individual GM events, recombinant DNA (r-DNA) segments, taxon-specific DNAs, and donor organisms of the respective r-DNAs. In this article, we report the single-laboratory validation of both DNA extraction methods and component PCR assays constituting the real-time PCR array. We selected some DNA extraction methods for specified plant matrixes, i.e., maize flour, soybean flour, and ground canola seeds, then evaluated the DNA quantity, DNA fragmentation, and PCR inhibition of the resultant DNA extracts. For the component PCR assays, we evaluated the specificity and LOD. All DNA extraction methods and component PCR assays satisfied the criteria set on the basis of previous reports. PMID:22649939

  2. Molecular detection and identification of intimin alleles in pathogenic Escherichia coli by multiplex PCR.

    PubMed

    Reid, S D; Betting, D J; Whittam, T S

    1999-08-01

    A multiplex PCR was designed to detect the eae gene and simultaneously identify specific alleles in pathogenic Escherichia coli. The method was tested on 87 strains representing the diarrheagenic E. coli clones. The results show that the PCR assay accurately detects eae and resolves alleles encoding the alpha, beta, and gamma intimin variants. PMID:10405431

  3. Molecular Detection and Identification of Intimin Alleles in Pathogenic Escherichia coli by Multiplex PCR

    PubMed Central

    Reid, Sean D.; Betting, David J.; Whittam, Thomas S.

    1999-01-01

    A multiplex PCR was designed to detect the eae gene and simultaneously identify specific alleles in pathogenic Escherichia coli. The method was tested on 87 strains representing the diarrheagenic E. coli clones. The results show that the PCR assay accurately detects eae and resolves alleles encoding the α, β, and γ intimin variants. PMID:10405431

  4. Detection of Toxoplasma gondii oocysts in water sample concentrates by real-time PCR

    Technology Transfer Automated Retrieval System (TEKTRAN)

    PCR techniques in combination with conventional parasite concentration procedures have potential for sensitive and specific detection of Toxoplasma gondii oocysts in water. Three real-time PCR assays based on the B1 gene and a 529-bp repetitive element were compared for detection of T. gondii tachyz...

  5. PCR-based Detection of Spiroplasma Citri Associated with Citrus Stubborn Disease

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Improvements in polymerase chain reaction (PCR) detection of Spiroplasma citri, the causal agent of citrus stubborn disease, made PCR more reliable than culturing for S. citri detection. Primer sequences from the P89 putative adhesin gene, which is present on a plasmid as well as in the S. citri gen...

  6. PCR-based Detection of Spiroplasma citri Associated with Citrus Stubborn Disease

    Technology Transfer Automated Retrieval System (TEKTRAN)

    PCR detection of Spiroplasma citri, the causal agent of citrus stubborn disease, was improved using primers based on sequences of the P89 adhesin gene and the P58 putative adhesin multigene of S. citri. PCR was compared with isolation by culturing for detection of S. citri in two 20 A citrus orchar...

  7. Quantitative real-time PCR (qPCR)--based tool for detection and quantification of Cordyceps militaris in soil.

    PubMed

    Saragih, Syaiful Amri; Takemoto, S; Hisamoto, Y; Fujii, M; Sato, H; Kamata, N

    2015-01-01

    A quantitative real-time PCR using a primer pair CM2946F/CM3160R was developed for specific detection and quantification of Cordyceps militaris from soil. Standard curves were obtained for genomic DNA and DNA extracts from autoclaved soil with a certain dose of C. militaris suspension. C. militaris was detected from two forest soil samples out of ten that were collected when fruit bodies of C. militaris were found. This method seemed effective in detection of C. militaris in the soil and useful for rapid and reliable quantification of C. militaris in different ecosystems. PMID:25446034

  8. Analytical Sensitivity Comparison between Singleplex Real-Time PCR and a Multiplex PCR Platform for Detecting Respiratory Viruses.

    PubMed

    Parker, Jayme; Fowler, Nisha; Walmsley, Mary Louise; Schmidt, Terri; Scharrer, Jason; Kowaleski, James; Grimes, Teresa; Hoyos, Shanann; Chen, Jack

    2015-01-01

    Multiplex PCR methods are attractive to clinical laboratories wanting to broaden their detection of respiratory viral pathogens in clinical specimens. However, multiplexed assays must be well optimized to retain or improve upon the analytic sensitivity of their singleplex counterparts. In this experiment, the lower limit of detection (LOD) of singleplex real-time PCR assays targeting respiratory viruses is compared to an equivalent panel on a multiplex PCR platform, the GenMark eSensor RVP. LODs were measured for each singleplex real-time PCR assay and expressed as the lowest copy number detected 95-100% of the time, depending on the assay. The GenMark eSensor RVP LODs were obtained by converting the TCID50/mL concentrations reported in the package insert to copies/μL using qPCR. Analytical sensitivity between the two methods varied from 1.2-1280.8 copies/μL (0.08-3.11 log differences) for all 12 assays compared. Assays targeting influenza A/H3N2, influenza A/H1N1pdm09, influenza B, and human parainfluenza 1 and 2 were most comparable (1.2-8.4 copies/μL, <1 log difference). Largest differences in LOD were demonstrated for assays targeting adenovirus group E, respiratory syncytial virus subtype A, and a generic assay for all influenza A viruses regardless of subtype (319.4-1280.8 copies/μL, 2.50-3.11 log difference). The multiplex PCR platform, the GenMark eSensor RVP, demonstrated improved analytical sensitivity for detecting influenza A/H3 viruses, influenza B virus, human parainfluenza virus 2, and human rhinovirus (1.6-94.8 copies/μL, 0.20-1.98 logs). Broader detection of influenza A/H3 viruses was demonstrated by the GenMark eSensor RVP. The relationship between TCID50/mL concentrations and the corresponding copy number related to various ATCC cultures is also reported. PMID:26569120

  9. Analytical Sensitivity Comparison between Singleplex Real-Time PCR and a Multiplex PCR Platform for Detecting Respiratory Viruses

    PubMed Central

    Parker, Jayme; Fowler, Nisha; Walmsley, Mary Louise; Schmidt, Terri; Scharrer, Jason; Kowaleski, James; Grimes, Teresa; Hoyos, Shanann; Chen, Jack

    2015-01-01

    Multiplex PCR methods are attractive to clinical laboratories wanting to broaden their detection of respiratory viral pathogens in clinical specimens. However, multiplexed assays must be well optimized to retain or improve upon the analytic sensitivity of their singleplex counterparts. In this experiment, the lower limit of detection (LOD) of singleplex real-time PCR assays targeting respiratory viruses is compared to an equivalent panel on a multiplex PCR platform, the GenMark eSensor RVP. LODs were measured for each singleplex real-time PCR assay and expressed as the lowest copy number detected 95–100% of the time, depending on the assay. The GenMark eSensor RVP LODs were obtained by converting the TCID50/mL concentrations reported in the package insert to copies/μL using qPCR. Analytical sensitivity between the two methods varied from 1.2–1280.8 copies/μL (0.08–3.11 log differences) for all 12 assays compared. Assays targeting influenza A/H3N2, influenza A/H1N1pdm09, influenza B, and human parainfluenza 1 and 2 were most comparable (1.2–8.4 copies/μL, <1 log difference). Largest differences in LOD were demonstrated for assays targeting adenovirus group E, respiratory syncytial virus subtype A, and a generic assay for all influenza A viruses regardless of subtype (319.4–1280.8 copies/μL, 2.50–3.11 log difference). The multiplex PCR platform, the GenMark eSensor RVP, demonstrated improved analytical sensitivity for detecting influenza A/H3 viruses, influenza B virus, human parainfluenza virus 2, and human rhinovirus (1.6–94.8 copies/μL, 0.20–1.98 logs). Broader detection of influenza A/H3 viruses was demonstrated by the GenMark eSensor RVP. The relationship between TCID50/mL concentrations and the corresponding copy number related to various ATCC cultures is also reported. PMID:26569120

  10. Improved detection of episomal Banana streak viruses by multiplex immunocapture PCR.

    PubMed

    Le Provost, Grégoire; Iskra-Caruana, Marie-Line; Acina, Isabelle; Teycheney, Pierre-Yves

    2006-10-01

    Banana streak viruses (BSV) are currently the main viral constraint to Musa germplasm movement, genetic improvement and mass propagation. Therefore, it is necessary to develop and implement BSV detection strategies that are both reliable and sensitive, such as PCR-based techniques. Unfortunately, BSV endogenous pararetrovirus sequences (BSV EPRVs) are present in the genome of Musa balbisiana. They interfere with PCR-based detection of episomal BSV in infected banana and plantain, such as immunocapture PCR. Therefore, a multiplex, immunocapture PCR (M-IC-PCR) was developed for the detection of BSV. Musa sequence tagged microsatellite site (STMS) primers were selected and used in combination with BSV species-specific primers in order to monitor possible contamination by Musa genomic DNA, using multiplex PCR. Furthermore, immunocapture conditions were optimized in order to prevent Musa DNA from interfering with episomal BSV DNA during the PCR step. This improved detection method successfully allowed the accurate, specific and sensitive detection of episomal DNA only from distinct BSV species. Its implementation should benefit PCR-based detection of viruses for which homologous sequences are present in the genome of their hosts, including transgenic plants expressing viral sequences. PMID:16857272

  11. Identification, Detection, and Enumeration of Human Bifidobacterium Species by PCR Targeting the Transaldolase Gene

    PubMed Central

    Requena, Teresa; Burton, Jeremy; Matsuki, Takahiro; Munro, Karen; Simon, Mary Alice; Tanaka, Ryuichiro; Watanabe, Koichi; Tannock, Gerald W.

    2002-01-01

    Methods that enabled the identification, detection, and enumeration of Bifidobacterium species by PCR targeting the transaldolase gene were tested. Bifidobacterial species isolated from the feces of human adults and babies were identified by PCR amplification of a 301-bp transaldolase gene sequence and comparison of the relative migrations of the DNA fragments in denaturing gradient gel electrophoresis (DGGE). Two subtypes of Bifidobacterium longum, five subtypes of Bifidobacterium adolescentis, and two subtypes of Bifidobacterium pseudocatenulatum could be differentiated using PCR-DGGE. Bifidobacterium angulatum and B. catenulatum type cultures could not be differentiated from each other. Bifidobacterial species were also detected directly in fecal samples by this combination of PCR and DGGE. The number of species detected was less than that detected by PCR using species-specific primers targeting 16S ribosomal DNA (rDNA). Real-time quantitative PCR targeting a 110-bp transaldolase gene sequence was used to enumerate bifidobacteria in fecal samples. Real-time quantitative PCR measurements of bifidobacteria in fecal samples from adults correlated well with results obtained by culture when either a 16S rDNA sequence or the transaldolase gene sequence was targeted. In the case of samples from infants, 16S rDNA-targeted PCR was superior to PCR targeting the transaldolase gene for the quantification of bifidobacterial populations. PMID:11976117

  12. Detection of feline calicivirus, feline herpesvirus 1 and Chlamydia psittaci mucosal swabs by multiplex RT-PCR/PCR.

    PubMed

    Sykes, J E; Allen, J L; Studdert, V P; Browning, G F

    2001-07-26

    A single tube, multiplex reverse transcription (RT)-polymerase chain reaction (PCR)/PCR assay was developed for detection of feline herpesvirus 1 (FHV1), Chlamydia psittaci and feline calicivirus (FCV) in cats with upper respiratory tract disease (URTD), incorporating a simple, rapid extraction procedure capable of extracting both DNA and RNA. The assay was found to be as sensitive in vitro as simplex assays that have previously been shown to be as sensitive as, or more sensitive than, culture for each pathogen in experimentally infected cats. Conjunctival alone or both conjunctival and oropharyngeal swabs were collected from cats in 104 households with URTD. FHV1 was detected in 18 (17.3%) and C. psittaci was detected in 12 (11.5%) households. The prevalence of C. psittaci was not significantly different to that determined using a duplex PCR assay for C. psittaci and FHV1. The prevalence of FCV was affected by sample storage temperature. Of samples stored at -70 degrees C, 0/31 were positive for FCV but FCV was detected in 10/73 (13.7%) samples stored at 4 degrees C (P=0.006). Of the samples stored at 4 degrees C, 3/19 (15.8%) conjunctival swabs were positive for FCV and 6/32 (18.8%) oropharyngeal/conjunctival swabs were positive for FCV (P=0.79). The potential utility of restriction endonuclease analysis of RT-PCR products resulting from amplification of the hypervariable region of the capsid protein gene of FCV in field samples, without prior cultivation, was also examined. The assay may have considerable importance for diagnosis and epidemiological surveys of feline upper respiratory tract pathogens. PMID:11376956

  13. Use of multiplex PCR and PCR restriction enzyme analysis for detection and exploration of the variability in the free-living amoeba Naegleria in the environment.

    PubMed

    Pélandakis, Michel; Pernin, Pierre

    2002-04-01

    A multiplex PCR was developed to simultaneously detect Naegleria fowleri and other Naegleria species in the environment. Multiplex PCR was also capable of identifying N. fowleri isolates with internal transcribed spacers of different sizes. In addition, restriction fragment length polymorphism analysis of the PCR product distinguished the main thermophilic Naegleria species from the sampling sites. PMID:11916734

  14. Use of Multiplex PCR and PCR Restriction Enzyme Analysis for Detection and Exploration of the Variability in the Free-Living Amoeba Naegleria in the Environment

    PubMed Central

    Pélandakis, Michel; Pernin, Pierre

    2002-01-01

    A multiplex PCR was developed to simultaneously detect Naegleria fowleri and other Naegleria species in the environment. Multiplex PCR was also capable of identifying N. fowleri isolates with internal transcribed spacers of different sizes. In addition, restriction fragment length polymorphism analysis of the PCR product distinguished the main thermophilic Naegleria species from the sampling sites. PMID:11916734

  15. HIGH SENSITIVE PCR METHOD FOR DETECTION OF PATHOGENIC Leptospira spp. IN PARAFFIN-EMBEDDED TISSUES

    PubMed Central

    Noda, Angel Alberto; Rodríguez, Islay; Rodríguez, Yaindrys; Govín, Anamays; Fernández, Carmen; Obregón, Ana Margarita

    2014-01-01

    This study describes the development and application of a new PCR assay for the specific detection of pathogenic leptospires and its comparison with a previously reported PCR protocol. New primers were designed for PCR optimization and evaluation in artificially-infected paraffin-embedded tissues. PCR was then applied to post-mortem, paraffin-embedded samples, followed by amplicon sequencing. The PCR was more efficient than the reported protocol, allowing the amplification of expected DNA fragment from the artificially infected samples and from 44% of the post-mortem samples. The sequences of PCR amplicons from different patients showed >99% homology with pathogenic leptospires DNA sequences. The applicability of a highly sensitive and specific tool to screen histological specimens for the detection of pathogenic Leptospira spp. would facilitate a better assessment of the prevalence and epidemiology of leptospirosis, which constitutes a health problem in many countries. PMID:25229221

  16. A PCR-ELISA for detecting Shiga toxin-producing Escherichia coli.

    PubMed

    Ge, Beilei; Zhao, Shaohua; Hall, Robert; Meng, Jianghong

    2002-03-01

    A sensitive and specific PCR-ELISA was developed to detect Escherichia coli O157:H7 and other Shiga toxin-producing E. coli (STEC) in food. The assay was based on the incorporation of digoxigenin-labeled dUTP and a biotin-labeled primer specific for Shiga toxin genes during PCR amplification. The labeled PCR products were bound to streptavidin-coated wells of a microtiter plate and detected by an ELISA. The specificity of the PCR was determined using 39 bacterial strains, including STEC, enteropathogenic E. coli, E. coli K12, and Salmonella. All of the STEC strains were positive, and non-STEC organisms were negative. The ELISA detecting system was able to increase the sensitivity of the PCR assay by up to 100-fold, compared with a conventional gel electrophoresis. The detection limit of the PCR-ELISA was 0.1-10 CFU dependent upon STEC serotypes, and genotypes of Shiga toxins. With the aid of a simple DNA extraction system, PrepMan, the PCR-ELISA was able to detect ca. 10(5) CFU of STEC per gram of ground beef without any culture enrichment. The entire procedure took about 6 h. Because of its microtiter plate format, PCR-ELISA is particularly suitable for large-scale screening and compatible with future automation. PMID:11909738

  17. Nested PCR for ultrasensitive detection of the potato ring rot bacterium, Clavibacter michiganensis subsp. sepedonicus.

    PubMed Central

    Lee, I M; Bartoszyk, I M; Gundersen, D E; Mogen, B; Davis, R E

    1997-01-01

    Oligonucleotide primers derived from sequences of the 16S rRNA gene (CMR16F1, CMR16R1, CMR16F2, and CMR16R2) and insertion element IS1121 of Clavibacter michiganensis subsp. sepedonicus (CMSIF1, CMSIR1, CMSIF2, and CMISR2) were used in nested PCR to detect the potato ring rot bacterium C. michiganensis subsp. sepedonicus. Nested PCR with primer pair CMSIF1-CMSIR1 followed by primer pair CMSIF2-CMSIR2 specifically detected C. michiganensis subsp. sepedonicus, while nested PCR with CMR16F1-CMR16R1 followed by CMR16F2-CMR16R2 detected C. michiganensis subsp. sepedonicus and the other C. michiganensis subspecies. In the latter case, C. michiganensis subsp. sepedonicus can be differentiated from the other subspecies by restriction fragment length polymorphism (RFLP) analyses of the nested PCR products (16S rDNA sequences). The nested PCR assays developed in this work allow ultrasensitive detection of very low titers of C. michiganensis subsp. sepedonicus which may be present in symptomiess potato plants or tubers and which cannot be readily detected by direct PCR (single PCR amplification). RFLP analysis of PCR products provides for an unambiguous confirmation of the identify of C. michiganensis subsp. sepedonicus. PMID:9212412

  18. Modified Real-Time PCR for Detecting, Differentiating, and Quantifying Ureaplasma urealyticum and Ureaplasma parvum

    PubMed Central

    Vancutsem, Ellen; Soetens, Oriane; Breugelmans, Maria; Foulon, Walter; Naessens, Anne

    2011-01-01

    We evaluated a previously described quantitative real-time PCR (qPCR) for quantifying and differentiating Ureaplasma parvum and U. urealyticum. Because of nonspecific reactions with Staphylococcus aureus DNA in the U. parvum PCR, we developed a modified qPCR and designed new primers. These oligonucleotides eradicated cross-reactions, indicating higher specificity. The detection limits of the qPCR were determined at 1 and 3 colony-forming units/ml for U. parvum and U. urealyticum, respectively. The quantification limits of the assay for both Ureaplasma species ranged from 2.106 to 2.101 copy numbers per PCR. A total of 300 patient samples obtained from the lower genital tract were tested with this newly designed qPCR assay and compared with culture results. Of the samples, 132 (44.0%) were culture positive, whereas 151 (50.3%) tested positive using qPCR. The U. parvum and U. urealyticum species were present in 79.5% and 12.6% of the qPCR-positive samples, respectively. Both species were found in 7.9% of those samples. Quantification of U. parvum and U. urealyticum in the samples ranged from less than 2.5 × 103 to 7.4 × 107 copies per specimen. In conclusion, the modified qPCR is a suitable method for rapid detection, differentiation, and quantification of U. parvum and U. urealyticum. PMID:21354056

  19. REAL-TIME PCR METHOD TO DETECT ENTEROCOCCUS FAECALIS IN WATER

    EPA Science Inventory

    A 16S rDNA real-time PCR method was developed to detect Enterococcus faecalis in water samples. The dynamic range for cell detection spanned five logs and the detection limit was determined to be 6 cfu/reaction. The assay was capable of detecting E. faecalis cells added to biof...

  20. A PCR procedure for the detection of Giardia intestinalis cysts and Escherichia coli in lettuce.

    PubMed

    Ramirez-Martinez, M L; Olmos-Ortiz, L M; Barajas-Mendiola, M A; Giono Cerezo, S; Avila, E E; Cuellar-Mata, P

    2015-06-01

    Giardia intestinalis is a pathogen associated with foodborne outbreaks and Escherichia coli is commonly used as a marker of faecal contamination. Implementation of routine identification methods of G. intestinalis is difficult for the analysis of vegetables and the microbiological detection of E. coli requires several days. This study proposes a PCR-based assay for the detection of E. coli and G. intestinalis cysts using crude DNA isolated from artificially contaminated lettuce. The G. intestinalis and E. coli PCR assays targeted the β-giardin and uidA genes, respectively, and were 100% specific. Forty lettuces from local markets were analysed by both PCR and light microscopy and no cysts were detected, the calculated detection limit was 20 cysts per gram of lettuce; however, by PCR, E. coli was detected in eight of ten randomly selected samples of lettuce. These data highlight the need to validate procedures for routine quality assurance. These PCR-based assays can be employed as alternative methods for the detection of G. intestinalis and E. coli and have the potential to allow for the automation and simultaneous detection of protozoa and bacterial pathogens in multiple samples. Significance and impact of the study: There are few studies for Giardia intestinalis detection in food because methods for its identification are difficult for routine implementation. Here, we developed a PCR-based method as an alternative to the direct observation of cysts in lettuce by light microscopy. Additionally, Escherichia coli was detected by PCR and the sanitary quality of lettuce was evaluated using molecular and standard microbiological methods. Using PCR, the detection probability of Giardia cysts inoculated onto samples of lettuce was improved compared to light microscopy, with the advantage of easy automation. These methods may be employed to perform timely and affordable detection of foodborne pathogens. PMID:25689035

  1. Acoustic detection of DNA conformation in genetic assays combined with PCR.

    PubMed

    Papadakis, G; Tsortos, A; Kordas, A; Tiniakou, I; Morou, E; Vontas, J; Kardassis, D; Gizeli, E

    2013-01-01

    Application of PCR to multiplexing assays is not trivial; it requires multiple fluorescent labels for amplicon detection and sophisticated software for data interpretation. Alternative PCR-free methods exploiting new concepts in nanotechnology exhibit high sensitivities but require multiple labeling and/or amplification steps. Here, we propose to simplify the problem of simultaneous analysis of multiple targets in genetic assays by detecting directly the conformation, rather than mass, of target amplicons produced in the same PCR reaction. The new methodology exploits acoustic wave devices which are shown to be able to characterize in a fully quantitative manner multiple double stranded DNAs of various lengths. The generic nature of the combined acoustic/PCR platform is shown using real samples and, specifically, during the detection of SNP genotyping in Anopheles gambiae and gene expression quantification in treated mice. The method possesses significant advantages to TaqMan assay and real-time PCR regarding multiplexing capability, speed, simplicity and cost. PMID:23778520

  2. Acoustic detection of DNA conformation in genetic assays combined with PCR

    PubMed Central

    Papadakis, G.; Tsortos, A.; Kordas, A.; Tiniakou, I.; Morou, E.; Vontas, J.; Kardassis, D.; Gizeli, E.

    2013-01-01

    Application of PCR to multiplexing assays is not trivial; it requires multiple fluorescent labels for amplicon detection and sophisticated software for data interpretation. Alternative PCR-free methods exploiting new concepts in nanotechnology exhibit high sensitivities but require multiple labeling and/or amplification steps. Here, we propose to simplify the problem of simultaneous analysis of multiple targets in genetic assays by detecting directly the conformation, rather than mass, of target amplicons produced in the same PCR reaction. The new methodology exploits acoustic wave devices which are shown to be able to characterize in a fully quantitative manner multiple double stranded DNAs of various lengths. The generic nature of the combined acoustic/PCR platform is shown using real samples and, specifically, during the detection of SNP genotyping in Anopheles gambiae and gene expression quantification in treated mice. The method possesses significant advantages to TaqMan assay and real-time PCR regarding multiplexing capability, speed, simplicity and cost. PMID:23778520

  3. Detection of sulfonamide resistance genes via in situ PCR-FISH.

    PubMed

    Gnida, Anna; Kunda, Katarzyna; Ziembińska, Aleksandra; Luczkiewicz, Aneta; Felis, Ewa; Surmacz-Górska, Joanna

    2014-01-01

    Due to the rising use of antibiotics and as a consequence of their concentration in the environment an increasing number of antibiotic resistant bacteria is observed. The phenomenon has a hazardous impact on human and animal life. Sulfamethoxazole is one of the sulfonamides commonly detected in surface waters and soil. The aim of the study was to detect sulfamethoxazole resistance genes in activated sludge biocenosis by use of in situ PCR and/or hybridization. So far no FISH probes for the detection of SMX resistance genes have been described in the literature. We have tested common PCR primers used for SMX resistance genes detection as FISH probes as well as a combination of in situ PCR and FISH. Despite the presence of SMX resistance genes in activated sludge confirmed via traditional PCR, the detection of the genes via microscopic visualization failed. PMID:25115110

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

    PubMed Central

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

    2016-01-01

    Background 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. Methodology/Principal Findings 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. Conclusions/Significance 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. PMID:26959642

  5. Detection of Entamoeba histolytica DNA in the Saliva of Amoebic Liver Abscess Patients Who Received Prior Treatment with Metronidazole

    PubMed Central

    Khairnar, Krishna; Parija, Subhash Chandra

    2008-01-01

    Saliva is an easily-accessible and a non-invasive clinical specimen alternate to blood and liver pus. An attempt was made to detect Entamoeba histolytica DNA released in the saliva of amoebic liver abscess (ALA) patients by applying 16S-like rRNA gene-based nested multiplex polymerase chain reaction (NM-PCR). The NM-PCR detected E. histolytica DNA in the saliva of eight (28.6%) of 28 ALA patients. The NM-PCR result was negative for E. histolytica DNA in the saliva of all the eight ALA patients who were tested prior to treatment with metronidazole but was positive in the saliva of eight (40%) of 20 ALA patient who were tested after therapy with metronidazole. The NM-PCR detected E. histolytica DNA in liver abscess pus of all 28 (100%) patients with ALA. The TechLab E. histolytica II enzyme-linked immunosorbent assay was positive for E. histolytica Gal/GalNAc lectin antigen in the liver abscess pus of 13 (46.4%) of the 28 ALA patients. The indirect haemagglutination (IHA) test was positive for anti-amoebic antibodies in the serum of 22 (78.6%) of the 28 ALA patients and 2 (5.7%) of 35 healthy controls. The present study, for the first time, demonstrates the release of E. histolytica DNA in the saliva of ALA patients by applying NM-PCR. PMID:19069620

  6. Detection of Entamoeba histolytica DNA in the saliva of amoebic liver abscess patients who received prior treatment with metronidazole.

    PubMed

    Khairnar, Krishna; Parija, Subhash Chandra

    2008-12-01

    Saliva is an easily-accessible and a non-invasive clinical specimen alternate to blood and liver pus. An attempt was made to detect Entamoeba histolytica DNA released in the saliva of amoebic liver abscess (ALA) patients by applying 16S-like rRNA gene-based nested multiplex polymerase chain reaction (NM-PCR). The NM-PCR detected E. histolytica DNA in the saliva of eight (28.6%) of 28 ALA patients. The NM-PCR result was negative for E. histolytica DNA in the saliva of all the eight ALA patients who were tested prior to treatment with metronidazole but was positive in the saliva of eight (40%) of 20 ALA patient who were tested after therapy with metronidazole. The NM-PCR detected E. histolytica DNA in liver abscess pus of all 28 (100%) patients with ALA. The TechLab E. histolytica II enzyme-linked immunosorbent assay was positive for E. histolytica Gal/GalNAc lectin antigen in the liver abscess pus of 13 (46.4%) of the 28 ALA patients. The indirect haemagglutination (IHA) test was positive for anti-amoebic antibodies in the serum of 22 (78.6%) of the 28 ALA patients and 2 (5.7%) of 35 healthy controls. The present study, for the first time, demonstrates the release of E. histolytica DNA in the saliva of ALA patients by applying NM-PCR. PMID:19069620

  7. An integrated microfluidic system for bovine DNA purification and digital PCR detection.

    PubMed

    Tian, Qingchang; Mu, Ying; Xu, Yanan; Song, Qi; Yu, Bingwen; Ma, Congcong; Jin, Wei; Jin, Qinhan

    2015-12-15

    In this paper, we described an integrated modularized microfluidic system that contained two distinct functional modules, one for nucleic acids (NA) extraction and the other for digital PCR (dPCR), allowing for detecting the bovine DNA in ovine tissue. PMID:26364950

  8. Ten hour real-time PCR technique for detection of Salmonella in meats

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We evaluated the efficacy of real-time PCR assays to detect low levels of Salmonella in meats following 8 h of pre-enrichment. The sensitivity and accuracy of molecular beacon and TaqMan probe PCR assays were compared with the conventional USDA microbiological procedure using artificially contaminat...

  9. Development of a real-time PCR assay for detection and quantification of Anaplasma ovis infection.

    PubMed

    Chi, Q; Liu, Z; Li, Y; Yang, J; Chen, Z; Yue, C; Luo, J; Yin, H

    2013-11-01

    Anaplasma ovis is a tick-borne intra-erythrocytic rickettsial pathogen of small ruminants. Real-time PCR possesses merits of rapidity, accuracy, reliability, automation and ease of standardization, but has not been used for detection of A. ovis, to the best of our knowledge. In this study, a real-time PCR assay was developed for detection and quantification of A. ovis. Species-specific primers and TaqMan probe were designed based on the gltA gene. No cross-reactions were observed with Anaplasma marginale, Anaplasma bovis, Anaplasma phagocytophilum, Borrelia burgdorferi s. l., Chlamydia psittaci, Mycoplasma mycoides, Theileria luwenshuni and Babesia sp. Xinjiang isolate. Analytic sensitivity results revealed that real-time PCR could detect as few as 10 copies of the gltA gene. The performance of real-time PCR was assessed by testing 254 blood samples from goats and comparing with the results from conventional PCR. This demonstrated that the real-time PCR assay was significantly more sensitive than conventional PCR. Our results indicated that real-time PCR is a useful approach for detecting A. ovis infections and has potential as an alternative tool for ecological and epidemiological surveillance of ovine anaplasmosis. PMID:24589111

  10. Low-cost, real-time, continuous flow PCR system for pathogen detection.

    PubMed

    Fernández-Carballo, B Leticia; McGuiness, Ian; McBeth, Christine; Kalashnikov, Maxim; Borrós, Salvador; Sharon, Andre; Sauer-Budge, Alexis F

    2016-04-01

    In this paper, we present a portable and low cost point-of-care (POC) PCR system for quantitative detection of pathogens. Our system is based on continuous flow PCR which maintains fixed temperatures zones and pushes the PCR solution between two heated areas allowing for faster heat transfer and as a result, a faster PCR. The PCR system is built around a 46.0 mm × 30.9 mm × 0.4 mm disposable thermoplastic chip. In order to make the single-use chip economically viable, it was manufactured by hot embossing and was designed to be compatible with roll-to-roll embossing for large scale production. The prototype instrumentation surrounding the chip includes two heaters, thermal sensors, and an optical system. The optical system allows for pathogen detection via real time fluorescence measurements. FAM probes were used as fluorescent reporters of the amplicons generated during the PCR. To demonstrate the function of the chip, two infectious bacteria targets were selected: Chlamydia trachomatis and Escherichia coli O157:H7. For both bacteria, the limit of detection of the system was determined, PCR efficiencies were calculated, and different flow velocities were tested. We have demonstrated successful detection for these two bacterial pathogens highlighting the versatility and broad utility of our portable, low-cost, and rapid PCR diagnostic device. PMID:26995085

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

    SciTech Connect

    Wang, Rong-Fu; Cao, Wei-Wen; Cerniglia, C.E.

    1996-04-01

    PCR procedures based on 16S rRNA genen sequence specific for 12 anaerobic bacteria that predominate in the human intestinal tract were developed and used for quantitative detection of these species in human feces and animal feces. The reported PCR procedure including the fecal sample preparation method is simplified and rapid and eliminates the DNA isolation steps.

  12. Single Laboratory Comparison of Quantitative Real-time PCR Assays for the Detection of Fecal Pollution

    EPA Science Inventory

    There are numerous quantitative real-time PCR (qPCR) assays available to detect and enumerate fecal pollution in ambient waters. Each assay employs distinct primers and probes that target different rRNA genes and microorganisms leading to potential variations in concentration es...

  13. Quantitative Detection of Listeria monocytogenes in Biofilms by Real-Time PCR

    PubMed Central

    Guilbaud, Morgan; de Coppet, Pierre; Bourion, Fabrice; Rachman, Cinta; Prévost, Hervé; Dousset, Xavier

    2005-01-01

    A quantitative method based on a real-time PCR assay to enumerate Listeria monocytogenes in biofilms was developed. The specificity for L. monocytogenes of primers targeting the listeriolysin gene was demonstrated using a SYBR Green I real-time PCR assay. The number of L. monocytogenes detected growing in biofilms was 6 × 102 CFU/cm2. PMID:15812058

  14. Comparative analysis of techniques for detection of quiescent Botrytis cinerea in grapes by quantitative PCR

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Quantitative PCR (qPCR) can be used to detect and monitor pathogen colonization, but early attempts to apply the technology to quiescent Botrytis cinerea infections of grape berries identified some specific limitations. In this study, four DNA extraction methods, two tissue-grinding methods, two gra...

  15. Type A influenza virus detection from horses by real-time RT-PCR and insulated isothermal RT-PCR.

    PubMed

    Balasuriya, Udeni B R

    2014-01-01

    Equine influenza (EI) is a highly contagious disease of horses caused by the equine influenza virus (EIV) H3N8 subtype. EI is the most important respiratory virus infection of horses and can disrupt major equestrian events and cause significant economic losses to the equine industry worldwide. Influenza H3N8 virus spreads rapidly in susceptible horses and can result in very high morbidity within 24-48 h after exposure to the virus. Therefore, rapid and accurate diagnosis of EI is critical for implementation of prevention and control measures to avoid the spread of EIV and to reduce the economic impact of the disease. The probe-based real-time reverse transcriptase polymerase chain reaction (rRT-PCR) assays targeting various EIV genes are reported to be highly sensitive and specific compared to the Directigen Flu A(®) test and virus isolation in embryonated hens' eggs. Recently, a TaqMan(®) probe-based insulated isothermal RT-PCR (iiRT-PCR) assay for the detection of EIV H3N8 subtype has been described. These molecular based diagnostic assays provide a fast and reliable means of EIV detection and disease surveillance. PMID:24899448

  16. A new nanoPCR molecular assay for detection of porcine bocavirus.

    PubMed

    Wang, Xiaoling; Bai, Aiquan; Zhang, Jing; Kong, Miaomiao; Cui, Yuchao; Ma, Xingjie; Ai, Xia; Tang, Qinghai; Cui, Shangjin

    2014-06-01

    Nanoparticle-assisted polymerase chain reaction (nanoPCR) is a novel method for the rapid amplification of DNA and has been used for the detection of virus. For detection of porcine bocavirus (PBoV), a sensitive and specific nanoPCR assay was developed with a pair of primers that were designed based on NS1 gene sequences available in GenBank. Under the optimized conditions of the PBoV nanoPCR assay, the nanoPCR assay was 100-fold more sensitive than a conventional PCR assay. The lower detection limit of the nanoPCR assay was about 6.70×10(1) copies. The nanoPCR assay amplified the specific 482-bp fragment of the PBoV NS1 recombinant plasmid but did not produce any product with genomic DNA or cDNA of porcine parvovirus, porcine circovirus type II, porcine reproductive and respiratory syndrome virus, pseudorabies virus, classic swine fever virus, Encephalomyocarditis virus, Porcine Teschovirus or African swine fever virus plasmid. Of 65 clinical samples collected from diseased pigs, 73.8% and 86.2% were determined to be PBoV positive by PBoV conventional PCR and PBoV nanoPCR assay, respectively. Of 36 clinical samples from healthy pigs, 27.8% and 44.4% were PBoV positive by PBoV conventional PCR and PBoV nanoPCR assay, respectively. The nanoPCR assay will be useful for diagnosing PBoV and for studying its epidemiology and pathology. PMID:24642242

  17. 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. PMID:10089280

  18. Nucleic acid extraction from polluted estuarine water for detection of viruses and bacteria by PCR and RT-PCR analysis.

    PubMed

    Petit, F; Craquelin, S; Guespin-Michel, J; Buffet-Janvresse, C

    1999-03-01

    We describe an extraction protocol for genomic DNA and RNA of both viruses and bacteria from polluted estuary water. This procedure was adapted to the molecular study of microflora of estuarine water where bacteria and viruses are found free, forming low-density biofilms, or intimately associated with organo-mineral particles. The sensitivity of the method was determined with seeded samples for RT-PCR and PCR analysis of viruses (10 virions/mL), and bacteria (1 colony-forming unit mL). We report an example of molecular detection of both poliovirus and Salmonella in the Seine estuary (France) and an approach to studying their association with organo-mineral particles. PMID:10209769

  19. Real-Time PCR: Revolutionizing Detection and Expression Analysis of Genes

    PubMed Central

    Deepak, SA; Kottapalli, KR; Rakwal, R; Oros, G; Rangappa, KS; Iwahashi, H; Masuo, Y; Agrawal, GK

    2007-01-01

    Invention of polymerase chain reaction (PCR) technology by Kary Mullis in 1984 gave birth to real-time PCR. Real-time PCRdetection and expression analysis of gene(s) in real-time — has revolutionized the 21st century biological science due to its tremendous application in quantitative genotyping, genetic variation of inter and intra organisms, early diagnosis of disease, forensic, to name a few. We comprehensively review various aspects of real-time PCR, including technological refinement and application in all scientific fields ranging from medical to environmental issues, and to plant. PMID:18645596

  20. Optimisation of DNA extraction and validation of PCR assays to detect Mycobacterium avium subsp. paratuberculosis.

    PubMed

    Timms, Verlaine J; Mitchell, Hazel M; Neilan, Brett A

    2015-05-01

    The aim of this study was to investigate DNA extraction methods and PCR assays suitable for the detection of Mycobacterium paratuberculosis in bovine tissue. The majority of methods currently used to detect M. paratuberculosis have been developed using bovine samples, such as faeces, blood or tissue and, in many cases, have been based on detection from pooled samples from a herd. However most studies have not compared PCR results to culture results. In order to address this problem, four DNA extraction protocols and three PCR assays were employed to detect M. paratuberculosis in bovine tissue. Given that culture is reliable from cows, the results were then compared with the known M. paratuberculosis culture status. The following DNA extractions were included, two commercial kits, a boiling method, an in house extraction based on a published method and enrichment by sonication. The three PCR assays used included single round IS900 and f57 assays and a nested IS900 assay. In addition, another PCR assay was validated for the detection of any Mycobacterial species and a universal bacterial 16S rRNA gene assay was used to detect sample inhibition. The in-house DNA extraction was the most consistent in extracting good quality DNA compared to all other methods. The use of two PCR markers, IS900 and f57, and a universal PCR enabled the correct samples to be identified as M. paratuberculosis positive. In addition, when compared to the culture result, false-positives did not occur and PCR inhibition was readily identified. Using an in house DNA extraction coupled with the IS900 and f57 PCR markers, this study provides a reliable and simple method to detect M. paratuberculosis in both veterinary and spill over infections. PMID:25797305

  1. Simultaneous Detection of Genetically Modified Organisms in a Mixture by Multiplex PCR-Chip Capillary Electrophoresis.

    PubMed

    Patwardhan, Supriya; Dasari, Srikanth; Bhagavatula, Krishna; Mueller, Steffen; Deepak, Saligrama Adavigowda; Ghosh, Sudip; Basak, Sanjay

    2015-01-01

    An efficient PCR-based method to trace genetically modified food and feed products is in demand due to regulatory requirements and contaminant issues in India. However, post-PCR detection with conventional methods has limited sensitivity in amplicon separation that is crucial in multiplexing. The study aimed to develop a sensitive post-PCR detection method by using PCR-chip capillary electrophoresis (PCR-CCE) to detect and identify specific genetically modified organisms in their genomic DNA mixture by targeting event-specific nucleotide sequences. Using the PCR-CCE approach, novel multiplex methods were developed to detect MON531 cotton, EH 92-527-1 potato, Bt176 maize, GT73 canola, or GA21 maize simultaneously when their genomic DNAs in mixtures were amplified using their primer mixture. The repeatability RSD (RSDr) of the peak migration time was 0.06 and 3.88% for the MON531 and Bt176, respectively. The RSD (RSDR) of the Cry1Ac peak ranged from 0.12 to 0.40% in multiplex methods. The method was sensitive in resolving amplicon of size difference up to 4 bp. The PCR-CCE method is suitable to detect multiple genetically modified events in a composite DNA sample by tagging their event specific sequences. PMID:26525256

  2. Direct Detection of Erythromycin-Resistant Bordetella pertussis in Clinical Specimens by PCR.

    PubMed

    Wang, Zengguo; Han, Ruijun; Liu, Ying; Du, Quanli; Liu, Jifeng; Ma, Chaofeng; Li, Hengxin; He, Qiushui; Yan, Yongping

    2015-11-01

    Resistance of Bordetella pertussis to erythromycin has been increasingly reported. We developed an allele-specific PCR method for rapid detection of erythromycin-resistant B. pertussis directly from nasopharyngeal (NP) swab samples submitted for diagnostic PCR. Based on the proven association of erythromycin resistance with the A2047G mutation in the 23S rRNA of B. pertussis, four primers, two of which were designed to be specific for either the wild-type or the mutant allele, were used in two different versions of the allele-specific PCR assay. The methods were verified with results obtained by PCR-based sequencing of 16 recent B. pertussis isolates and 100 NP swab samples submitted for diagnostic PCR. The detection limits of the two PCR assays ranged from 10 to 100 fg per reaction for both erythromycin-susceptible and -resistant B. pertussis. Two amplified fragments of each PCR, of 286 and 112 bp, respectively, were obtained from a mutant allele of the isolates and/or NP swab samples containing B. pertussis DNAs. For the wild-type allele, only a 286-bp fragment was visible when the allele-specific PCR assay 1 was performed. No amplification was found when a number of non-Bordetella bacterial pathogens and NP swab samples that did not contain the DNAs of B. pertussis were examined. This assay can serve as an alternative for PCR-based sequencing, especially for local laboratories in resource-poor countries. PMID:26224847

  3. Integrated sorting, concentration and real time PCR based detection system for sensitive detection of microorganisms

    PubMed Central

    Nayak, Monalisha; Singh, Deepak; Singh, Himanshu; Kant, Rishi; Gupta, Ankur; Pandey, Shashank Shekhar; Mandal, Swarnasri; Ramanathan, Gurunath; Bhattacharya, Shantanu

    2013-01-01

    The extremely low limit of detection (LOD) posed by global food and water safety standards necessitates the need to perform a rapid process of integrated detection with high specificity, sensitivity and repeatability. The work reported in this article shows a microchip platform which carries out an ensemble of protocols which are otherwise carried in a molecular biology laboratory to achieve the global safety standards. The various steps in the microchip include pre-concentration of specific microorganisms from samples and a highly specific real time molecular identification utilizing a q-PCR process. The microchip process utilizes a high sensitivity antibody based recognition and an electric field mediated capture enabling an overall low LOD. The whole process of counting, sorting and molecular identification is performed in less than 4 hours for highly dilute samples. PMID:24253282

  4. Integrated sorting, concentration and real time PCR based detection system for sensitive detection of microorganisms

    NASA Astrophysics Data System (ADS)

    Nayak, Monalisha; Singh, Deepak; Singh, Himanshu; Kant, Rishi; Gupta, Ankur; Pandey, Shashank Shekhar; Mandal, Swarnasri; Ramanathan, Gurunath; Bhattacharya, Shantanu

    2013-11-01

    The extremely low limit of detection (LOD) posed by global food and water safety standards necessitates the need to perform a rapid process of integrated detection with high specificity, sensitivity and repeatability. The work reported in this article shows a microchip platform which carries out an ensemble of protocols which are otherwise carried in a molecular biology laboratory to achieve the global safety standards. The various steps in the microchip include pre-concentration of specific microorganisms from samples and a highly specific real time molecular identification utilizing a q-PCR process. The microchip process utilizes a high sensitivity antibody based recognition and an electric field mediated capture enabling an overall low LOD. The whole process of counting, sorting and molecular identification is performed in less than 4 hours for highly dilute samples.

  5. Rapid detection and identification of Clostridium chauvoei by PCR based on flagellin gene sequence.

    PubMed

    Kojima, A; Uchida, I; Sekizaki, T; Sasaki, Y; Ogikubo, Y; Tamura, Y

    2001-02-26

    We developed a one-step polymerase chain reaction (PCR) system that specifically detects Clostridium chauvoei. Oligonucleotide primers were designed to amplify a 516-bp fragment of the structural flagellin gene. The specificity of the PCR was investigated by analyzing 59 strains of clostridia, and seven strain of other genera. A 516-bp fragment could be amplified from all the C. chauvoei strains tested, and no amplification was observed by using DNAs from the other strains tested, including Clostridium septicum. Similarly, this PCR-based method specifically detected C. chauvoei DNA sequences in samples of muscle and exudate of obtained from mice within 12h of inoculation. In tests using samples of muscle or liver, the limit of detection was about 200 organisms per reaction. These results suggest that the one-step PCR system may be useful for direct detection and identification of C. chauvoei in clinical specimens. PMID:11182502

  6. DETECTION AND IDENTIFICATION OF PATHOGENIC CANDIDA SPECIES IN WATER USING FLOW CYTOMETRY COUPLED WITH TAQMAN PCR

    EPA Science Inventory

    As the incidence of human fungal infection increases, the ability to detect and identify pathogenic fungi in potential environmental reservoirs becomes increasingly important for disease control. PCR based assays are widely used for diagnostic purposes, but may be inadequate for...

  7. Detection and identification of infectious bronchitis virus by RT-PCR in Iran.

    PubMed

    Homayounimehr, Alireza; Pakbin, Ahmad; Momayyez, Reza; Fatemi, Seyyedeh Mahsa Rastegar

    2016-06-01

    Infectious bronchitis virus (IBV) causes severe diseases in poultry with significant economic consequences to the poultry industry in Iran. The aim of this study was the detection and identification of IBV by reverse transcription(RT)-PCR in Iran. Ten IB virus strains were detected by testing trachea, cecal tonsil, and kidney tissues collected from broiler and layer farms in Iran. In order to detect infectious bronchitis virus, an optimized RT-PCR was used. Primers targeting the conserved region of known IBV serotypes were used in the RT-PCR assay. Primers selectively detecting Massachusetts and 793/B type IB viruses were designed to amplify the S1 gene of the virus and used in the nested PCR test. Our findings indicate the circulation of at least three genotypes of IB viruses (Massachusetts, 793/B, and variant 2) among poultry flocks. PMID:27010714

  8. Development of multiplex PCR assay for simultaneous detection of five bacterial fish pathogens.

    PubMed

    Altinok, Ilhan; Capkin, Erol; Kayis, Sevki

    2008-10-15

    A multiplex polymerase chain reaction (PCR) method was designed for the simultaneous detection of the five major fish pathogens, Aeromonas hydrophila, Aeromonas salmonicida subsp. salmonicida, Flavobacterium columnare, Renibacterium salmoninarum, and Yersinia ruckeri. Each of the five pairs of oligonucleotide primers exclusively amplified the targeted gene of the specific microorganism. The detection limits of the multiplex PCR was in the range of 2, 1, 1, 3, and 1CFU for A. hydrophila, A. salmonicida, F. columnare, R. salmoninarum, and Y. ruckeri, respectively. Multiplex PCR did not produce any nonspecific amplification products when tested against 23 related species of bacteria. The multiplex PCR assay was useful for the detection of the bacteria in naturally infected fish. This assay is a sensitive and specific and reproducible diagnostic tool for the simultaneous detection of five pathogenic bacteria that cause disease in fish. Therefore, it could be a useful alternative to the conventional culture based method. PMID:18499358

  9. 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. PMID:26210470

  10. Detection of Methicillin-Resistant Staphylococcus aureus by a Duplex Droplet Digital PCR Assay

    PubMed Central

    Kelley, KaShonda; Cosman, Angela; Belgrader, Phillip; Chapman, Brenda

    2013-01-01

    Health care-associated infections with methicillin-resistant Staphylococcus aureus (MRSA) contribute to significant hospitalization costs. We report here a droplet digital PCR (ddPCR) assay, which is a next-generation emulsion-based endpoint PCR assay for high-precision MRSA analysis. Reference cultures of MRSA, methicillin-susceptible S. aureus (MSSA), and confounders were included as controls. Copan swabs were used to sample cultures and collect specimens for analysis from patients at a large teaching hospital. Swab extraction and cell lysis were accomplished using magnetic-driven agitation of silica beads. Quantitative PCR (qPCR) (Roche Light Cycler 480) and ddPCR (Bio-Rad QX100 droplet digital PCR system) assays were used to detect genes for the staphylococcal protein SA0140 (SA) and the methicillin resistance (mecA) gene employing standard TaqMan chemistries. Both qPCR and ddPCR assays correctly identified culture controls for MRSA (76), MSSA (12), and confounder organisms (36) with 100% sensitivity and specificity. Analysis of the clinical samples (211 negative and 186 positive) collected during a study of MRSA nasal carriage allowed direct comparison of the qPCR and ddPCR assays to the Cepheid MRSA GeneXpert assay. A total of 397 clinical samples were examined in this study. Cepheid MRSA GeneXpert values were used to define negative and positive samples. Both the qPCR and ddPCR assays were in good agreement with the reference assay. The sensitivities for the qPCR and ddPCR assays were 96.8% (95% confidence interval [CI], 93.1 to 98.5%) and 96.8% (95% CI, 93.1 to 98.5%), respectively. Both the qPCR and ddPCR assays had specificities of 91.9% (95% CI, 87.5 to 94.9%) for qPCR and 91.0% (95% CI, 86.4 to 94.2%) for ddPCR technology. PMID:23596244

  11. One-PCR-tube approach for in situ DNA isolation and detection.

    PubMed

    Huang, Xin; Hou, Lihua; Xu, Xiaohe; Chen, Hongjun; Ji, Haifeng; Zhu, Shuifang

    2011-10-21

    Traditional real-time polymerase chain reaction (PCR) requires a purified DNA sample for PCR amplification and detection. This requires PCR tests be conducted in clean laboratories, and limits its applications for field tests. This work developed a method that can carry out DNA purification, amplification and detection in a single PCR tube. The polypropylene PCR tube was first treated with chromic acid and peptide nucleic acids (PNA) as DNA-capturer were immobilized on the internal surface of the tube. Cauliflower mosaic virus 35S (CaMV-35S) promoter in the crude extract was hybridized with the PNA on the tube surface, and the inhibitors, interfering agents and irrelevant DNA in the crude extract were effectively removed by rinsing with buffer solutions. The tube that has captured the target DNA can be used for the following real-time PCR (RT-PCR). By using this approach, the detection of less than 2500 copies of 35S plasmids in a complex sample could be completed within 3 hours. Chocolate samples were tested for real sample analysis, and 35S plasmids in genetically modified chocolate samples have been successfully identified with this method in situ. The novel One-PCR-tube method is competitive for commercial kits with the same time and simpler operation procedure. This method may be widely used for identifying food that contains modified DNA and specific pathogens in the field. PMID:21879029

  12. Comparison of PCR and Plaque Assay for Detection and Enumeration of Coliphage in Polluted Marine Waters

    PubMed Central

    Rose, J. B.; Zhou, X.; Griffin, D. W.; Paul, J. H.

    1997-01-01

    A total of 68 marine samples from various sites impacted by sewage and storm waters were analyzed by both the plaque assay and a reverse transcriptase (RT) PCR technique for F(sup+)-specific coliphage. The coliphage levels detected by the plaque assay averaged 1.90 x 10(sup4) PFU/100.0 ml. Using a most probable number (MPN) PCR approach, the levels averaged 2.40 x 10(sup6) MPN-PCR units/100.0 ml. Two samples were positive by RT-PCR but negative by plaque assay, and 12 samples were positive by plaque assay but negative by RT-PCR (levels lower than 11.00 PFU/100.0 ml). The host system used for the plaque assay may detect somatic coliphage in addition to the F(sup+)-specific coliphage. When it is used as an indicator of pollution, contamination may be missed with more restrictive systems. The difference in results may be due to the sensitivity, specificity, or inhibition of RT-PCR in marine samples. This study provides information on quantifying PCR results by an MPN method and insights into interpretation of PCR data for detection of viruses in marine environments. PMID:16535737

  13. High throughput multiplex PCR and probe-based detection with Luminex beads for seven intestinal parasites.

    PubMed

    Taniuchi, Mami; Verweij, Jaco J; Noor, Zannatun; Sobuz, Shihab U; Lieshout, Lisette van; Petri, William A; Haque, Rashidul; Houpt, Eric R

    2011-02-01

    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

  14. A multiplex PCR for the simultaneous detection of Tenacibaculum maritimum and Edwardsiella tarda in aquaculture.

    PubMed

    Castro, Nuria; Toranzo, Alicia E; Magariños, Beatriz

    2014-06-01

    A specific and sensitive multiplex PCR (mPCR) method was developed as a useful tool for the simultaneous detection of two important flatfish pathogens in marine aquaculture, Tenacibaculum maritimum and Edwardsiella tarda. In fish tissues, the average detection limit for these mPCR-amplified organisms was 2 × 10 ⁵ ± 0.2 CFU/g and 4 × 10 ⁵ ± 0.3 CFU/g, respectively. These values are similar or even lower than those previously obtained using the corresponding single PCR. Moreover, mPCR did not produce any nonspecific amplification products when tested against 36 taxonomically related and unrelated strains belonging to 33 different bacterial species. Large amounts of DNA from one of the target bacterial species in the presence of low amounts from the other did not have a significant effect on the amplification sensitivity of the latter. PMID:26418855

  15. PCR for detection of Clostridium botulinum type C in avian and environmental samples.

    PubMed

    Franciosa, G; Fenicia, L; Caldiani, C; Aureli, P

    1996-04-01

    A PCR was developed and applied for the detection of Clostridium botulinum type C in 18 avian and environmental samples collected during an outbreak of avian botulism, and the results were compared with those obtained by conventional methodologies based on the mouse bioassay. PCR and mouse bioassay results compared well (100%) after the enrichment of samples, but PCR results directly indicated the presence of this microorganism in six samples, while only one of these contained the type C botulinal neurotoxin before enrichment. The PCR assay was sensitive (limit of detection between 15 and 15 x 10(3) spores per PCR), specific (no amplification products were obtained with other clostridia), and rapid, since sonicated and heated samples provided enough template for amplification without any DNA purification. Eleven isolates of C. botulinum type C were recovered from mallards (Anas platyrhynchos), grey herons (Ardea cinerea), and mud during investigation of this outbreak. PMID:8815101

  16. PCR for detection of Clostridium botulinum type C in avian and environmental samples.

    PubMed Central

    Franciosa, G; Fenicia, L; Caldiani, C; Aureli, P

    1996-01-01

    A PCR was developed and applied for the detection of Clostridium botulinum type C in 18 avian and environmental samples collected during an outbreak of avian botulism, and the results were compared with those obtained by conventional methodologies based on the mouse bioassay. PCR and mouse bioassay results compared well (100%) after the enrichment of samples, but PCR results directly indicated the presence of this microorganism in six samples, while only one of these contained the type C botulinal neurotoxin before enrichment. The PCR assay was sensitive (limit of detection between 15 and 15 x 10(3) spores per PCR), specific (no amplification products were obtained with other clostridia), and rapid, since sonicated and heated samples provided enough template for amplification without any DNA purification. Eleven isolates of C. botulinum type C were recovered from mallards (Anas platyrhynchos), grey herons (Ardea cinerea), and mud during investigation of this outbreak. PMID:8815101

  17. Detecting Newcastle disease virus in combination of RT-PCR with red blood cell absorption.

    PubMed

    Yi, Jianzhong; Liu, Chengqian

    2011-01-01

    Reverse transcription-polymerase chain reaction (RT-PCR) has limited sensitivity when treating complicated samples, such as feces, waste-water in farms, and nucleic acids, protein rich tissue samples, all the factors may interfere with the sensitivity of PCR test or generate false results. In this study, we developed a sensitive RT-PCR, combination of red blood cell adsorption, for detecting Newcastle disease virus (NDV). One pair of primers which was highly homologous to three NDV pathotypes was designed according to the consensus nucleocapsid protein (NP) gene sequence. To eliminate the interfere of microbes and toxic substances, we concentrated and purified NDV from varied samples utilizing the ability of NDV binding red blood cells (RBCs). The RT-PCR coupled with red blood cell adsorption was much more sensitive in comparison with regular RT-PCR. The approach could also be used to detect other viruses with the property of hemagglutination, such as influenza viruses. PMID:21535888

  18. 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

  19. Detection of Leptospira DNA in Patients with Aseptic Meningitis by PCR

    PubMed Central

    Romero, Eliete C.; Billerbeck, Ana E. C.; Lando, Valéria S.; Camargo, Eide D.; Souza, Candida C.; Yasuda, Paulo H.

    1998-01-01

    Samples of cerebrospinal fluid from 103 patients with aseptic meningitis were tested by PCR for detection of leptospires, and the results were compared with those of the microscopic agglutination test (MAT) and an enzyme-linked immunosorbent assay for detection of immunoglobulin M (ELISA-IgM). Of these samples, 39.80% were positive by PCR and 8.74 and 3.88% were positive by MAT and ELISA-IgM, respectively. PMID:9574730

  20. [Selective detection of viable pathogenic bacteria in water using reverse transcription quantitative PCR].

    PubMed

    Lin, Yi-Wen; Li, Dan; Wu, Shu-Xu; He, Miao; Yang, Tian

    2012-11-01

    A reverse transcription q quantitative PCR (RT-qPCR) assay method was established, which can quantify the copy numbers of RNA in pathogenic bacteria of E. coli and Enterococcus faecium. The results showed that cDNA was generated with the RT-PCR reagents, target gene was quantified with the qPCR, the copy numbers of RNA were stable at about 1 copies x CFU(-1) for E. coli and 7.98 x 10(2) copies x CFU(-1) for Enterococcus faecium respectively during the stationary grow phase for the both indicator bacteria [E. coli (6-18 h) and Enterococcus faecium (10-38 h)]. The established RT-qPCR method can quantify the numbers of viable bacteria through detecting bacterial RNA targets. Through detecting the heat-treated E. coli and Enterococcus faecium by three methods (culture method, qPCR, RT-qPCR), we found that the qPCR and RT-qPCR can distinguish 1.43 lg copy non-viable E. coli and 2.5 lg copy non-viable Enterococcus faecium. These results indicated that the established methods could effectively distinguish viable bacteria from non-viable bacteria. Finally we used this method to evaluate the real effluents of the secondary sedimentation of wastewater treatment plant (WWTP), the results showed that the correlation coefficients (R2) between RT-qPCR and culture method were 0.930 (E. coli) and 0.948 (Enterococcus faecium), and this established RT-PCR method can rapidly detect viable pathogenic bacteria in genuine waters. PMID:23323443

  1. Real-Time PCR Improves Helicobacter pylori Detection in Patients with Peptic Ulcer Bleeding

    PubMed Central

    Casalots, Alex; Sanfeliu, Esther; Boix, Loreto; García-Iglesias, Pilar; Sánchez-Delgado, Jordi; Montserrat, Antònia; Bella-Cueto, Maria Rosa; Gallach, Marta; Sanfeliu, Isabel; Segura, Ferran; Calvet, Xavier

    2011-01-01

    Background and Aims Histological and rapid urease tests to detect H. pylori in biopsy specimens obtained during peptic ulcer bleeding episodes (PUB) often produce false-negative results. We aimed to examine whether immunohistochemistry and real-time PCR can improve the sensitivity of these biopsies. Patients and Methods We selected 52 histology-negative formalin-fixed paraffin-embedded biopsy specimens obtained during PUB episodes. Additional tests showed 10 were true negatives and 42 were false negatives. We also selected 17 histology-positive biopsy specimens obtained during PUB to use as controls. We performed immunohistochemistry staining and real-time PCR for 16S rRNA, ureA, and 23S rRNA for H. pylori genes on all specimens. Results All controls were positive for H. pylori on all PCR assays and immunohistochemical staining. Regarding the 52 initially negative biopsies, all PCR tests were significantly more sensitive than immunohistochemical staining (p<0.01). Sensitivity and specificity were 55% and 80% for 16S rRNA PCR, 43% and 90% for ureA PCR, 41% and 80% for 23S rRNA PCR, and 7% and 100% for immunohistochemical staining, respectively. Combined analysis of PCR assays for two genes were significantly more sensitive than ureA or 23S rRNA PCR tests alone (p<0.05) and marginally better than 16S rRNA PCR alone. The best combination was 16S rRNA+ureA, with a sensitivity of 64% and a specificity of 80%. Conclusions Real-time PCR improves the detection of H. pylori infection in histology-negative formalin-fixed paraffin-embedded biopsy samples obtained during PUB episodes. The low reported prevalence of H. pylori in PUB may be due to the failure of conventional tests to detect infection. PMID:21625499

  2. Real-Time PCR Method for Detection of Pathogenic Yersinia enterocolitica in Food▿

    PubMed Central

    Lambertz, S. Thisted; Nilsson, C.; Hallanvuo, S.; Lindblad, M.

    2008-01-01

    The current methods for the detection of pathogenic Yersinia enterocolitica bacteria in food are time consuming and inefficient. Therefore, we have developed and evaluated in-house a TaqMan probe-based real-time PCR method for the detection of this pathogen. The complete method comprises overnight enrichment, DNA extraction, and real-time PCR amplification. Also included in the method is an internal amplification control. The selected primer-probe set was designed to use a 163-bp amplicon from the chromosomally located gene ail (attachment and invasion locus). The selectivity of the PCR method was tested with a diverse range (n = 152) of related and unrelated strains, and no false-negative or false-positive PCR results were obtained. The sensitivity of the PCR amplification was 85 fg purified genomic DNA, equivalent to 10 cells per PCR tube. Following the enrichment of 10 g of various food samples (milk, minced beef, cold-smoked sausage, fish, and carrots), the sensitivity ranged from 0.5 to 55 CFU Y. enterocolitica. Good precision, robustness, and efficiency of the PCR amplification were also established. In addition, the method was tested on naturally contaminated food; in all, 18 out of 125 samples were positive for the ail gene. Since no conventional culture method could be used as a reference method, the PCR products amplified from these samples were positively verified by using conventional PCR and sequencing of the amplicons. A rapid and specific real-time PCR method for the detection of pathogenic Y. enterocolitica bacteria in food, as presented here, provides a superior alternative to the currently available detection methods and makes it possible to identify the foods at risk for Y. enterocolitica contamination. PMID:18708521

  3. Rapid detection of Salmonella in bovine lymph nodes using a commercial real-time PCR system

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Rapid Salmonella detection is needed to help prevent the distribution of contaminated food products. Using traditional culture methods, Salmonella detection can take up to 3-5 days. Using an improved protocol and a commercial real-time PCR system, we have shortened the detection time to under 24 h...

  4. Multiplex RT-PCR detection of H3N2 influenza A virus in dogs.

    PubMed

    Lee, EunJung; Kim, Eun-Ju; Kim, Bo-Hye; Song, Jae-Young; Cho, In-Soo; Shin, Yeun-Kyung

    2016-02-01

    A multiplex RT-PCR (mRT-PCR) assay to detect H3N2 CIV genomic segments was developed as a rapid and cost-effective method. Its performance was evaluated with forty-six influenza A viruses from different hosts using three primer sets which amplify four segments of H3N2 CIV simultaneously. The mRT-PCR has been successful in detecting the viral segments, indicating that it can improve the speed of diagnosis for H3N2 CIV and its reassortants. PMID:26738688

  5. Detection of Verotoxigenic Escherichia coli by Magnetic Capture-Hybridization PCR

    PubMed Central

    Chen, Jinru; Johnson, Roger; Griffiths, Mansel

    1998-01-01

    Magnetic capture-hybridization PCR (MCH-PCR) was used for the detection of 36 verotoxigenic (verotoxin [VT]-producing) Escherichia coli (VTEC), 5 VTEC reference, and 13 non-VTEC control cultures. The detection system employs biotin-labeled probes to capture the DNA segments that contain specific regions of the genes for VT1 and VT2 by DNA-DNA hybridization. The hybrids formed were isolated by streptavidin-coated magnetic beads which were collected by a magnetic particle separator and, subsequently, amplified directly by conventional PCR. The detection system was found to be specific for VTEC: no amplification was obtained from non-VTEC controls, whereas VTEC isolates tested positive for one or two specific PCR products. With 5, 7, or 10 h of enrichment, the limits of detection were 103, 102, and 100 CFU/ml, respectively, by agarose gel electrophoresis. Southern hybridization did not seem to improve the limit of the detection. When applied to food, MCH-PCR was capable of detecting 100 CFU of VTEC per g of ground beef with 15 h of nonselective enrichment. The results of MCH-PCR for pure cultures of VT1- and/or VT2-producing E. coli cells were in total agreement with toxin production as measured by a VT enzyme-linked immunosorbent assay. PMID:9435072

  6. 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. PMID:26839086

  7. Heminested PCR assay for detection of six genotypes of rabies and rabies-related viruses.

    PubMed Central

    Heaton, P R; Johnstone, P; McElhinney, L M; Cowley, R; O'Sullivan, E; Whitby, J E

    1997-01-01

    A heminested reverse transcriptase PCR (hnRT-PCR) protocol which is rapid and sensitive for the detection of rabies virus and rabies-related viruses is described. Sixty isolates from six of the seven genotypes of rabies and rabies-related viruses were screened successfully by hnRT-PCR and Southern blot hybridization. Of the 60 isolates, 93% (56 of 60) were positive by external PCR, while all isolates were detected by heminested PCR and Southern blot hybridization. We also report on a comparison of the sensitivity of the standard fluorescent-antibody test (FAT) for rabies antigen and that of hnRT-PCR for rabies viral RNA with degraded tissue infected with a genotype 1 virus. Results indicated that FAT failed to detect viral antigen in brain tissue that was incubated at 37 degrees C for greater than 72 h, while hnRT-PCR detected viral RNA in brain tissue that was incubated at 37 degrees C for 360 h. PMID:9350729

  8. Ultrasensitive Detection of RNA and DNA Viruses Simultaneously Using Duplex UNDP-PCR Assay

    PubMed Central

    Wang, Zengguo; Zhang, Xiujuan; Zhao, Xiaomin; Du, Qian; Chang, Lingling; Tong, Dewen

    2015-01-01

    Mixed infection of multiple viruses is common in modern intensive pig rearing. However, there are no methods available to detect DNA and RNA viruses in the same reaction system in preclinical level. In this study, we aimed to develop a duplex ultrasensitive nanoparticle DNA probe-based PCR assay (duplex UNDP-PCR) that was able to simultaneously detect DNA and RNA viruses in the same reaction system. PCV2 and TGEV are selected as representatives of the two different types of viruses. PCV2 DNA and TGEV RNA were simultaneously released from the serum sample by boiling with lysis buffer, then magnetic beads and gold nanoparticles coated with single and/or duplex specific probes for TGEV and PCV2 were added to form a sandwich-like complex with nucleic acids released from viruses. After magnetic separation, DNA barcodes specific for PCV2 and TGEV were eluted using DTT and characterized by specific PCR assay for specific DNA barcodes subsequently. The duplex UNDP-PCR showed similar sensitivity as that of single UNDP-PCR and was able to detect 20 copies each of PCV2 and TGEV in the serum, showing approximately 250-fold more sensitivity than conventional duplex PCR/RT-PCR assays. No cross-reaction was observed with other viruses. The positive detection rate of single MMPs- and duplex MMPs-based duplex UNDP-PCR was identical, with 29.6% for PCV2, 9.3% for TGEV and 3.7% for PCV2 and TGEV mixed infection. This duplex UNDP-PCR assay could detect TGEV (RNA virus) and PCV2 (DNA virus) from large-scale serum samples simultaneously without the need for DNA/RNA extraction, purification and reverse transcription of RNA, and showed a significantly increased positive detection rate for PCV2 (29%) and TGEV (11.7%) preclinical infection than conventional duplex PCR/RT-PCR. Therefore, the established duplex UNDP-PCR is a rapid and economical detection method, exhibiting high sensitivity, specificity and reproducibility. PMID:26544710

  9. DNA Sequence Signatures for Rapid Detection of Six Target Bacterial Pathogens Using PCR Assays

    PubMed Central

    Nagamine, Kenjiro; Hung, Guo-Chiuan; Li, Bingjie; Lo, Shyh-Ching

    2015-01-01

    Using Streptococcus pyogenes as a model, we previously established a stepwise computational workflow to effectively identify species-specific DNA signatures that could be used as PCR primer sets to detect target bacteria with high specificity and sensitivity. In this study, we extended the workflow for the rapid development of PCR assays targeting Enterococcus faecalis, Enterococcus faecium, Clostridium perfringens, Clostridium difficile, Clostridium tetani, and Staphylococcus aureus, which are of safety concern for human tissue intended for transplantation. Twenty-one primer sets that had sensitivity of detecting 5–50 fg DNA from target bacteria with high specificity were selected. These selected primer sets can be used in a PCR array for detecting target bacteria with high sensitivity and specificity. The workflow could be widely applicable for the rapid development of PCR-based assays for a wide range of target bacteria, including those of biothreat agents. PMID:26279626

  10. DNA Sequence Signatures for Rapid Detection of Six Target Bacterial Pathogens Using PCR Assays.

    PubMed

    Nagamine, Kenjiro; Hung, Guo-Chiuan; Li, Bingjie; Lo, Shyh-Ching

    2015-01-01

    Using Streptococcus pyogenes as a model, we previously established a stepwise computational workflow to effectively identify species-specific DNA signatures that could be used as PCR primer sets to detect target bacteria with high specificity and sensitivity. In this study, we extended the workflow for the rapid development of PCR assays targeting Enterococcus faecalis, Enterococcus faecium, Clostridium perfringens, Clostridium difficile, Clostridium tetani, and Staphylococcus aureus, which are of safety concern for human tissue intended for transplantation. Twenty-one primer sets that had sensitivity of detecting 5-50 fg DNA from target bacteria with high specificity were selected. These selected primer sets can be used in a PCR array for detecting target bacteria with high sensitivity and specificity. The workflow could be widely applicable for the rapid development of PCR-based assays for a wide range of target bacteria, including those of biothreat agents. PMID:26279626

  11. Immuno-PCR: Very sensitive antigen detection by means of specific antibody-DNA conjugates

    SciTech Connect

    Sano, T.; Smith, C.L.; Cantor, C.R. )

    1992-10-02

    An antigen detection system, termed immuno-polymerase chain reaction (immuno-PCR), was developed in which a specific DNA molecule is used as the marker. A streptavidin-protein A chimera that possesses tight and specific binding affinity both for biotin and immunoglobulin G was used to attach a biotinylated DNA specifically to antigen-monoclonal antibody complexes that had been immobilized on microtiter plate wells. Then, a segment of the attached DNA was amplified by PCR. Analysis of the PCR products by agarose gel electrophoresis after staining with ethidium bromide allowed as few as 580 antigen molecules to be readily and reproducibly detected. Direct comparison with enzyme-linked immunosorbent assay with the use of a chimera-alkaline phosphatase conjugate demonstrates that enhancement in detection sensitivity was obtained with the use of immuno-PCR. Given the enormous amplification capability and specificity of PCR, this immuno-PCR technology has a sensitivity greater than any existing antigen detection system and, in principle, could be applied to the detection of single antigen molecules.

  12. Development of a nested-PCR assay for the detection of Cryptosporidium parvum in finished water.

    PubMed

    Monis, P T; Saint, C P

    2001-05-01

    A nested-PCR assay, incorporating an internal positive control, was developed for Cryptosporidium monitoring in finished water. This assay was capable of reproducibly detecting 8 oocysts in spiked-filtered water samples collected from 5 South Australian water treatment plants. The RT-PCR assay of Kaucner and Stinear (Appl. Environ. Microbiol. 64(5) (1998) 1743) was also evaluated for the detection of Cryptosporidium parvum. Initially, under our experimental conditions, a detection level of 27 oocysts was achieved for spiked reagent water samples. This level was improved to 5 oocysts by modification of the method. Untreated South Australian source waters concentrated by calcium carbonate flocculation were found to be highly inhibitory to the RT-PCR assay. Concentration of similar samples using Envirochek filters appeared to eliminate PCR inhibition. While both methods possessed similar sensitivities the nested-PCR assay was more reproducible, more cost effective, simpler to perform and could detect both viable and non-viable intact Cryptosporidium parvum oocysts, which is an important consideration for plant operators. These factors make the nested-PCR assay the method of choice for screening large numbers of potable water samples, where a reliable low level of detection is essential. PMID:11329665

  13. Accuracy of Reverse Dot-Blot PCR in Detection of Different β-Globin Gene Mutations.

    PubMed

    El-Fadaly, N; Abd-Elhameed, A; Abd-Elbar, E; El-Shanshory, M

    2016-06-01

    Prevention programs for β-thalassemia based on molecular diagnosis of heterozygous carriers and/or patients require the use of reliable mutation screening methods. The aim of this study was to compare between direct DNA sequencing, and reverse dot-blot PCR in detection of different β-globin gene mutations in Egyptian children with β-thalassemia. Forty children with β-thalassemia were subjected to mutation analysis, performed by both direct DNA sequencing and β-globin Strip Assay MED™ (based on reverse dot-blot PCR). The most frequent mutant alleles detected by reverse dot-blot PCR were; IVSI-110 G>A (31.25 %), IVS I-6 T > C (21.25 %), and IVS I-1 G>A (20 %). Relatively less frequent mutant alleles detected by reverse dot-blot PCR were "IVSII-1 G>A (5 %), IVSII-745 C>G (5 %), IVSII-848 C>A (2.5 %), IVSI-5 G>C (2.5 %), -87 C>G(2.5 %), and cd39 C>T (2.5 %)", While the genotypes of three patients (6 alleles 7.5 %) were not detected by reverse dot-blot PCR. Mutant alleles detected by direct DNA sequencing were the same as reverse dot-blot PCR method except it revealed the genotypes of 3 undetected patients (one patient was homozygous IVSI-110 G>A, and two patients were homozygous IVS I-1 G>A. Sensitivity of the reverse dot-blot PCR was 92.5 % when compared to direct DNA sequencing for detecting β-thalassemia mutations. Our results therefore suggest that, direct DNA sequencing may be preferred over reverse dot-blot PCR in critical diagnostic situations like genetic counseling for prenatal diagnosis. PMID:27065589

  14. 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

  15. Detection of Legionella species in environmental water by the quantitative PCR method in combination with ethidium monoazide treatment.

    PubMed

    Inoue, Hiroaki; Takama, Tomoko; Yoshizaki, Miwa; Agata, Kunio

    2015-01-01

    We detected Legionella species in 111 bath water samples and 95 cooling tower water samples by using a combination of conventional plate culture, quantitative polymerase chain reaction (qPCR) and qPCR combined with ethidium monoazide treatment (EMA-qPCR) methods. In the case of bath water samples, Legionella spp. were detected in 30 samples by plate culture, in 85 samples by qPCR, and in 49 samples by EMA-qPCR. Of 81 samples determined to be Legionella-negative by plate culture, 56 and 23 samples were positive by qPCR and EMA-qPCR, respectively. Therefore, EMA treatment decreased the number of Legionella-positive bath water samples detected by qPCR. In contrast, EMA treatment had no effect on cooling tower water samples. We therefore expect that EMA-qPCR is a useful method for the rapid detection of viable Legionella spp. from bath water samples. PMID:25817816

  16. Duplex PCR for detection of Salmonella and Shigella spp in cockle samples.

    PubMed

    Senachai, Pachara; Chomvarin, Chariya; Wongboot, Warawan; Boonyanugomol, Wongwarut; Tangkanakul, Waraluk

    2013-09-01

    Salmonella and Shigella spp are important causative agents of foodborne diseases. A sensitive, specific and rapid method is essential for detection of these pathogens. In this study, a duplex PCR method was developed for simultaneous detection of Salmonella and Shigella spp in cockle samples and compared with the traditional culture method. Enrichment broths for Salmonella spp recovery were also compared. Sensitivity of the duplex PCR for simultaneous detection of Salmonella and Shigella spp from pure culture was 10(3) CFU/ml (40 CFU/PCR reaction), and that of sterile cockle samples spiked with these two pathogens was 1 CFU/10 g of cockle tissue after 9 hours enrichment [3 hours in buffered peptone water (BPW), followed by 6 hours in Rappaport Vasiliadis (RV) broth or tetrathionate (TT) broth for Salmonella spp and 6 hours enrichment in Shigella broth (SB) for Shigella spp]. There was no significant difference in detection sensitivity between enrichment in RV and TT broths. Salmonella spp detected in cockles in Khon Kaen, Thailand by duplex PCR and culture method was 17% and 13%, respectively but Shigella spp was not detected. The duplex PCR technique developed for simultaneous detection of Salmonella and Shigella spp in cockle samples was highly sensitive, specific and rapid and could serve as a suitable method for food safety assessment. PMID:24437322

  17. Duplex PCR Methods for the Molecular Detection of Escherichia fergusonii Isolates from Broiler Chickens

    PubMed Central

    Simmons, Karen; Rempel, Heidi; Block, Glenn; Forgetta, Vincenzo; Vaillancourt, Rolland; Malouin, François; Topp, Edward; Delaquis, Pascal

    2014-01-01

    Escherichia fergusonii is an emerging pathogen that has been isolated from a wide range of infections in animals and humans. Primers targeting specific genes, including yliE (encoding a conserved hypothetical protein of the cellulose synthase and regulator of cellulose synthase island), EFER_1569 (encoding a hypothetical protein, putative transcriptional activator for multiple antibiotic resistance), and EFER_3126 (encoding a putative triphosphoribosyl-dephospho-coenzyme A [CoA]), were designed for the detection of E. fergusonii by conventional and real-time PCR methods. Primers were screened by in silico PCR against 489 bacterial genomic sequences and by both PCR methods on 55 reference and field strains. Both methods were specific and sensitive for E. fergusonii, showing amplification only for this bacterium. Conventional PCR required a minimum bacterial concentration of approximately 102 CFU/ml, while real-time PCR required a minimum of 0.3 pg of DNA for consistent detection. Standard curves showed an efficiency of 98.5%, with an R2 value of 0.99 for the real-time PCR assay. Cecal and cloacal contents from 580 chickens were sampled from broiler farms located in the Fraser Valley (British Columbia, Canada). Presumptive E. fergusonii isolates were recovered by enrichment and plating on differential and selective media. Of 301 total presumptive isolates, 140 (46.5%) were identified as E. fergusonii by biochemical profiling with the API 20E system and 268 (89.0%) using PCR methods. E. fergusonii detection directly from cecal and cloacal samples without preenrichment was achieved with both PCR methods. Hence, the PCR methods developed in this work significantly improve the detection of E. fergusonii. PMID:24441160

  18. The development of a real-time PCR to detect pathogenic Leptospira species in kidney tissue.

    PubMed

    Fearnley, C; Wakeley, P R; Gallego-Beltran, J; Dalley, C; Williamson, S; Gaudie, C; Woodward, M J

    2008-08-01

    A LightCycler real-time PCR hybridization probe-based assay that detects a conserved region of the16S rRNA gene of pathogenic but not saprophytic Leptospira species was developed for the rapid detection of pathogenic leptospires directly from processed tissue samples. In addition, a differential PCR specific for saprophytic leptospires and a control PCR targeting the porcine beta-actin gene were developed. To assess the suitability of these PCR methods for diagnosis, a trial was performed on kidneys taken from adult pigs with evidence of leptospiral infection, primarily a history of reproductive disease and serological evidence of exposure to pathogenic leptospires (n=180) and aborted pig foetuses (n=24). Leptospire DNA was detected by the 'pathogenic' specific PCR in 25 tissues (14%) and the control beta-actin PCR was positive in all 204 samples confirming DNA was extracted from all samples. No leptospires were isolated from these samples by culture and no positives were detected with the 'saprophytic' PCR. In a subsidiary experiment, the 'pathogenic' PCR was used to analyse kidney samples from rodents (n=7) collected as part of vermin control in a zoo, with show animals with high microagglutination titres to Leptospira species, and five were positive. Fifteen PCR amplicons from 1 mouse, 2 rat and 14 pig kidney samples, were selected at random from positive PCRs (n=30) and sequenced. Sequence data indicated L. interrogans DNA in the pig and rat samples and L. inadai DNA, which is considered of intermediate pathogenicity, in the mouse sample. The only successful culture was from this mouse kidney and the isolate was confirmed to be L. inadai by classical serology. These data suggest this suite of PCRs is suitable for testing for the presence of pathogenic leptospires in pig herds where abortions and infertility occur and potentially in other animals such as rodents. PMID:17961617

  19. Panfungal PCR Assay for Detection of Fungal Infection in Human Blood Specimens

    PubMed Central

    Van Burik, Jo-Anne; Myerson, David; Schreckhise, Randall W.; Bowden, Raleigh A.

    1998-01-01

    A novel panfungal PCR assay which detects the small-subunit rRNA gene sequence of the two major fungal organism groups was used to test whole-blood specimens obtained from a series of blood or bone marrow transplant recipients. The 580-bp PCR product was identified after amplification by panfungal primers and hybridization to a 245-bp digoxigenin-labeled probe. The lower limit of detection of the assay was approximately four organisms per milliliter of blood. Multiple whole-blood specimens from five patients without fungal infection or colonization had negative PCR results. Specimens from 11 infected patients had positive PCR results. Blood from three patients with pulmonary aspergillosis had positive PCR results: one patient’s blood specimen obtained in the week prior to the diagnosis of infection by a positive bronchoalveolar lavage fluid culture result was positive by PCR, and blood specimens obtained from two patients 1 to 2 days after lung biopsy and which were sterile by culture were positive by PCR. The blood of four patients with candidemia, three patients with mixed fungal infections, and one patient with fusariosis also had positive PCR signals. The panfungal PCR assay can detect multiple fungal genera and may be used as an adjunct to conventional methods for the detection of fungal infection or for describing the natural history of fungal infection. Further studies are needed to define the sensitivity and specificity of this assay for the diagnosis of fungal infection prior to the existence of other clinical or laboratory indications of invasive fungal infection. PMID:9574670

  20. Dielectrophoresis chips improve PCR detection of the food-spoiling yeast Zygosaccharomyces rouxii in apple juice.

    PubMed

    del Carmen Jaramillo, Maria; Huttener, Mario; Alvarez, Juan Manuel; Homs-Corbera, Antoni; Samitier, Josep; Torrents, Eduard; Juárez, Antonio

    2015-07-01

    Dielectrophoretic (DEP) manipulation of cells present in real samples is challenging. We show in this work that an interdigitated DEP chip can be used to trap and wash a population of the food-spoiling yeast Zygosaccharomyces rouxii that contaminates a sample of apple juice. By previously calibrating the chip, the yeast population loaded is efficiently trapped, washed, and recovered in a small-volume fraction that, in turn, can be used for efficient PCR detection of this yeast. DEP washing of yeast cells gets rid of PCR inhibitors present in apple juice and facilitates PCR analysis. This and previous works on the use of DEP chips to improve PCR analysis show that a potential use of DEP is to be used as a treatment of real samples prior to PCR. PMID:25808673

  1. Coagulase gene polymorphisms detected by PCR in Staphylococcus aureus isolated from subclinical bovine mastitis in Turkey.

    PubMed

    Karahan, Murat; Cetinkaya, Burhan

    2007-09-01

    The genetic relatedness of coagulase (coa) positive Staphylococcus aureus isolated from cows with subclinical mastitis in Turkey was investigated by polymerase chain reaction (PCR)-based restriction fragment length polymorphism (RFLP) analysis. Among 700 milk samples positive in the California Mastitis Test (CMT), species specific PCR identified 200 (28.6%) isolates as S. aureus and 161 (80.5%) of these isolates were positive for the 3' end of the coa gene by PCR. Most isolates (n=135, 83.9%) produced a single band on coa PCR, with molecular sizes ranging from 500 to 1400bp, whereas a small number of isolates (n=26, 16.1%) yielded two amplification products. Coa RFLP analysis using AluI and Hin6I revealed 23 and 22 band patterns, respectively. The detection of double bands by coa PCR, previously reported in human isolates, suggests that milking personnel can play a role in the transmission of S. aureus. PMID:16901735

  2. Development of a novel detection system for microbes from bovine diarrhea by real-time PCR

    PubMed Central

    TSUCHIAKA, Shinobu; MASUDA, Tsuneyuki; SUGIMURA, Satoshi; KOBAYASHI, Suguru; KOMATSU, Natsumi; NAGAI, Makoto; OMATSU, Tsutomu; FURUYA, Tetsuya; OBA, Mami; KATAYAMA, Yukie; KANDA, Shuhei; YOKOYAMA, Tadashi; MIZUTANI, Tetsuya

    2015-01-01

    Diarrhea in cattle is one of the most economically costly disorders, decreasing milk production and weight gain. In the present study, we established a novel simultaneous detection system using TaqMan real-time PCR designed as a system for detection of microbes from bovine diarrhea using real-time PCR (referred to as Dembo-PCR). Dembo-PCR simultaneously detects a total of 19 diarrhea-causing pathogens, including viruses, bacteria and protozoa. Specific primer–probe sets were newly designed for 7 pathogens and were synthesized on the basis of previous reports for 12 pathogens. Assays were optimized to react under the same reaction conditions. The PCR efficiency and correlation coefficient (R2) of standard curves for each assay were more than 80% and 0.9766, respectively. Furthermore, the sensitivity of Dembo-PCR in fecal sample analysis was measured with feces spiked with target pathogens or synthesized DNA that included specific nucleotide target regions. The resulting limits of detection (LOD) for virus-spiked samples, bacteria and DNA fragments were 0.16–1.6 TCID50 (PFU/reaction), 1.3–13 CFU/reaction and 10–100 copies/reaction, respectively. All reactions showed high sensitivity in pathogen detection. A total of 8 fecal samples, collected from 6 diarrheic cattle, 1 diarrheic calf and 1 healthy cow, were tested using Dembo-PCR to validate the assay’s clinical performance. The results revealed that bovine coronavirus had infected all diarrheic adult cattle and that bovine torovirus had infected the diarrheic calf. These results suggest that Dembo-PCR may be a powerful tool for diagnosing infectious agents in cattle diarrhea. PMID:26616156

  3. Standardized positive controls for detection of norovirus by reverse transcription PCR

    PubMed Central

    2011-01-01

    Background Norovirus is one of the most common causes of nonbacterial gastroenteritis in humans. Rapid spread by contaminated food and person-to-person transmission through the fecal-oral route are characteristics of norovirus epidemiology and result in high morbidity in vulnerable patient populations. Therefore, detection of norovirus is a major public health concern. Currently, the most common method for detecting and differentiating among norovirus strains in clinical and environmental samples is reverse transcription PCR (RT-PCR). Standardized positive controls used in RT-PCR assays to detect norovirus are designed to overcome the problem of false-negative results due to PCR inhibitors and suboptimal reaction conditions. Results In the current study, four types of RNA transcripts were produced from plasmids: norovirus GI-5 and GII-4 capsid regions with human rotavirus (VP7 gene derived) fragment insertions, and norovirus GI-6 and GII-4 capsid regions with hepatitis A virus (VP1/P2A gene derived) fragment insertions. These size-distinguishable products were used as positive controls under the RT-PCR assay conditions used to detect NoV in stool and groundwater samples. Their reliability and reproducibility was confirmed by multiple sets of experiments. Conclusions These standardized products may contribute to the reliable and accurate diagnosis by RT-PCR of norovirus outbreaks, when conducted by laboratories located in different regions. PMID:21612660

  4. Development and evaluation of a rapid and simple procedure for detection of Pneumocystis carinii by PCR.

    PubMed Central

    Cartwright, C P; Nelson, N A; Gill, V J

    1994-01-01

    We report the development of a simplified PCR-based assay for the detection of Pneumocystis carinii DNA in clinical specimens. The adoption of a rapid DNA extraction procedure and the introduction of a type of enzyme-linked immunosorbent assay for PCR product detection enabled this procedure to be carried out in a single working day in a clinical microbiology laboratory. The PCR assay was prospectively compared with an immunofluorescent-antibody (FA) staining method for the detection of P. carinii in induced sputum and bronchoalveolar lavage (BAL) specimens. The results of the study showed that, for induced sputum specimens, FA staining had a sensitivity of 78% (32 of 41 specimens) and a specificity of 100% (166 of 166 specimens); PCR was 100% (41 of 41 specimens) sensitive and 98% (162 of 166 specimens) specific. For BAL specimens, FA staining was 100% sensitive (21 of 21 specimens) and 100% specific (133 of 133 specimens), and PCR had a sensitivity of 100% (21 of 21 specimens) and a specificity of 99% (132 of 133 specimens). These results strongly suggest that use of our PCR-based assay could effect clinically useful improvements in the sensitivity of induced sputum specimens for the detection of P. carinii. Images PMID:7929749

  5. PCR and non-isotopic labeling techniques for plant virus detection.

    PubMed

    Fenby, N S; Scott, N W; Slater, A; Elliott, M C

    1995-07-01

    PCR technology permits the detection of viruses at levels several orders of magnitude lower than is possible by other methods. This high sensitivity facilitates detection of virus sequences during the early stages of infection of plants and in soil and vector samples. Early detection of beet necrotic yellow vein virus (BNYVV) in Beta vulgaris is an important part of the strategy for prevention of the spread of rhizomania, a commercially significant disease of sugar beet. A diagnostic test for BNYVV has been developed. This test involves amplification of the viral genome by PCR coupled with non-isotopic labeling and detection of specific sequences. The PCR amplification of BNYVV sequences has been optimized with respect to primer design, sample preparation and reaction conditions. Several non-isotopic labeling strategies for signal amplification have been compared. Hybridization with digoxigenin-labelled cDNA permits the most sensitive detection of PCR products and is the most appropriate method for routine diagnosis. These observations are discussed in the context of the application of PCR for detecting a wide range of viruses. PMID:7580844

  6. High sensitive method detection of plant RNA viruses by electrochemiluminescence reverse transcription PCR

    NASA Astrophysics Data System (ADS)

    Tang, Ya-bing; Xing, Da; Zhu, De-bin; Zhou, Xiao-ming

    2007-05-01

    It is well known that plant and animal viruses had widely spread the whole of world, and made a big loss in farming and husbandry. It is necessary that a highly efficient and accurate virus's detection method was developed. This research combines reverse transcription polymerase chain reaction (RT-PCR) technique with electrochemiluminescence method, to detect plant RNA viruses for the first time. Biotin-probe hybridizes with PCR product to specific select the target for detection, thus can avoid pseudo-positive result. TBR-probe hybridizes with PCR product to emit light for ECL detection. Specific nucleic acid sequences (20bp) were added to 5' terminal all of the primers, which can improve the chance of hybridization between TBR-probe and PCR product. At the same time, one of the PCR product chain can hybridize two Ru-probes, the ECL signal is intensified. The method was used to detect Odntoglossum ringspot virus ORSV, Sugarcane mosaic virus ScMV, Sorghum mosaic virus SrMV, and Maize dwarf mosaic virus MDMV, the experiment results show that this method could reliably identity virus infected plant samples. In a word, this method has higher sensitivity and lower cost than others. It can effectively detect the plant viruses with simplicity, stability, and high sensitivity.

  7. Development of Multiplex PCR for Simultaneous Detection of Three Pathogenic Shigella Species

    PubMed Central

    RANJBAR, Reza; AFSHAR, Davoud; MEHRABI TAVANA, Ali; NAJAFI, Ali; POURALI, Fatemeh; SAFIRI, Zahra; SOROURI ZANJANI, Rahim; JONAIDI JAFARI, Nematollah

    2014-01-01

    Background: Shigella species are among the common causes of bacterial diarrhoeal diseases. Traditional detection methods are time-consuming resulting in delay in treatment and control of Shigella infections thus there is a need to develop molecular methods for rapid and simultaneous detection of Shigella spp. In this study a rapid multiplex PCR were developed for simultaneous detection of three pathogenic Shigella species. Methods: For detection of Shigella spp., a pair of primers was used to replicate a chromosomal sequence. Three other sets of primers were also designed to amplify the target genes of three most common species of Shigella in Iran including S. sonnei, S. flexneri and S. boydii. The multiplex PCR assay was optimized for simultaneous detection and differentiation of three pathogenic Shigella species. The assay specificity was investigated by testing different strains of Shigella and other additional strains belonging to non Shigella species, but responsible for foodborne diseases. Results: The Shigella genus specific PCR yielded the expected DNA band of 159 bp in all tested strains belonging to four Shigella species. The standard and multiplex PCR assays also produced the expected fragments of 248 bp, 503 bp, and 314 bp, for S. boydii, S. sonnei and S. flexneri, respectively. Each species-specific primer pair did not show any cross-reactivity. Conclusion: Both standard and multiplex PCR protocols had a good specificity. They can provide a valuable tool for the rapid and simultaneous detection and differentiation of three most prevalent Shigella species in Iran. PMID:26171358

  8. Optimization of Quantitative PCR Methods for Enteropathogen Detection

    PubMed Central

    Liu, Jie; Gratz, Jean; Amour, Caroline; Nshama, Rosemary; Walongo, Thomas; Maro, Athanasia; Mduma, Esto; Platts-Mills, James; Boisen, Nadia; Nataro, James; Haverstick, Doris M.; Kabir, Furqan; Lertsethtakarn, Paphavee; Silapong, Sasikorn; Jeamwattanalert, Pimmada; Bodhidatta, Ladaporn; Mason, Carl; Begum, Sharmin; Haque, Rashidul; Praharaj, Ira; Kang, Gagandeep; Houpt, Eric R.

    2016-01-01

    Detection and quantification of enteropathogens in stool specimens is useful for diagnosing the cause of diarrhea but is technically challenging. Here we evaluate several important determinants of quantification: specimen collection, nucleic acid extraction, and extraction and amplification efficiency. First, we evaluate the molecular detection and quantification of pathogens in rectal swabs versus stool, using paired flocked rectal swabs and whole stool collected from 129 children hospitalized with diarrhea in Tanzania. Swabs generally yielded a higher quantification cycle (Cq) (average 29.7, standard deviation 3.5 vs. 25.3 ± 2.9 from stool, P<0.001) but were still able to detect 80% of pathogens with a Cq < 30 in stool. Second, a simplified total nucleic acid (TNA) extraction procedure was compared to separate DNA and RNA extractions and showed 92% (318/344) sensitivity and 98% (951/968) specificity, with no difference in Cq value for the positive results (ΔCq(DNA+RNA-TNA) = -0.01 ± 1.17, P = 0.972, N = 318). Third, we devised a quantification scheme that adjusts pathogen quantity to the specimen’s extraction and amplification efficiency, and show that this better estimates the quantity of spiked specimens than the raw target Cq. In sum, these methods for enteropathogen quantification, stool sample collection, and nucleic acid extraction will be useful for laboratories studying enteric disease. PMID:27336160

  9. Development of a multiplex PCR assay to detect gastroenteric pathogens in the feces of Mexican children.

    PubMed

    Tolentino-Ruiz, R; Montoya-Varela, D; García-Espitia, M; Salas-Benito, M; Gutiérrez-Escolano, A; Gómez-García, C; Figueroa-Arredondo, P; Salas-Benito, J; De Nova-Ocampo, M

    2012-10-01

    Acute gastroenteritis (AGE) is a major cause of childhood morbidity and mortality worldwide; the etiology of AGE includes viruses, bacteria, and parasites. A multiplex PCR assay to simultaneously identify human Astrovirus (HAstV), Calicivirus (HuCVs), Entamoeba histolytica (E. histolytica), and enteroinvasive Escherichia coli (EIEC) in stool samples is described. A total of 103 samples were individually analyzed by ELISA (enzyme-linked immunosorbent assays) and RT-PCR/PCR. HAstV and HuCVs were detected in four out of 103 samples (3.8 %) by RT-PCR, but ELISAs found only one sample as positive for HuCVs (2.5 %). E. histolytica was identified in two out of 19 samples (10.5 %) and EIEC in 13 out of 20 samples (70 %) by PCR, and all PCR products were sequenced to verify their identities. Our multiplex PCR results demonstrate the simultaneous amplification of different pathogens such as HAstV, EIEC, and E. histolytica in the same reaction, though the HuCVs signal was weak in every replicate. Regardless, this multiplex PCR protocol represents a novel tool for the identification of distinct pathogens and may provide support for the diagnosis of AGE in children. PMID:22711331

  10. Targeted PCR for Detection of Vaginal Bacteria Associated with Bacterial Vaginosis▿

    PubMed Central

    Fredricks, David N.; Fiedler, Tina L.; Thomas, Katherine K.; Oakley, Brian B.; Marrazzo, Jeanne M.

    2007-01-01

    Several novel bacterial species have been detected in subjects with bacterial vaginosis (BV) by using broad-range PCR assays, but this approach is insensitive for detecting minority species. We developed a series of taxon-directed 16S rRNA gene PCR assays for more sensitive detection of key vaginal bacteria. We sought to determine the prevalence of each species in the vagina, its association with BV, and the utility of PCR for the microbiological diagnosis of BV. Targeted PCR assays were developed for 17 vaginal bacterial species and applied to 264 vaginal-fluid samples from 81 subjects with and 183 subjects without BV. The results were compared to those of two widely accepted methods for diagnosing BV, the use of clinical findings (Amsel criteria) and the interpretation of vaginal-fluid Gram stains (Nugent criteria). Leptotrichia/Sneathia, Atopobium vaginae, an Eggerthella-like bacterium, Megasphaera species, and three novel bacteria in the order Clostridiales are among the bacterial species significantly associated with BV. PCR detection of either a Megasphaera species or one of the Clostridiales bacteria yielded a sensitivity of 99% and a specificity of 89% for diagnosis of BV compared to the Amsel clinical criteria and a sensitivity of 95.9% and a specificity of 93.7% compared to the Nugent criteria (Gram stain). PCR detection of one or more fastidious bacterial species is a more reliable indicator of BV than detection of bacteria, such as Gardnerella vaginalis, previously linked to BV, highlighting the potential of PCR for the diagnosis of BV. PMID:17687006

  11. Real-time RT-PCR Assay for Detection and Differentiation of Citrus Tristeza Virus Isolates

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Multiplex one step real time RT-PCR assays using TaqMan probes were developed for detection and strain differentiation of Citrus tristeza virus (CTV). For broad spectrum CTV detection, a TaqMan primer and Cy5-labeled probe were designed using CP gene sequences. An internal control was developed us...

  12. Signal Amplification by Glyco-qPCR for Ultrasensitive Detection of Carbohydrates: Applications in Glycobiology**

    PubMed Central

    Kwon, Seok Joon; Lee, Kyung Bok; Solakyildirim, Kemal; Masuko, Sayaka; Ly, Mellisa; Zhang, Fuming; Li, Lingyun; Dordick, Jonathan S.; Linhardt, Robert J.

    2012-01-01

    Tiny amounts of carbohydrates (ca. 1 zmol) can be detected quantitatively by a real-time method based on the conjugation of carbohydrates with DNA markers (see picture). The proposed method (glyco-qPCR) provides uniform, ultrasensitive detection of carbohydrates, which can be applied to glycobiology, as well as carbohydrate-based drug discovery. PMID:23073897

  13. Real-time RT-PCR assay for detection and differentiation of Citrus tristeza virus isolates

    Technology Transfer Automated Retrieval System (TEKTRAN)

    For universal detection of Citrus tristeza virus (CTV) strains by real time RT-PCR, a protocol was developed based on a set of primers and a Cy5-labeled TaqMan probe. This test included primers and a TET-labeled TaqMan probe selected on the mitochondrial nad5 gene for the simultaneous detection of ...

  14. Detection of infectious enteroviruses by an integrated cell culture-PCR procedure.

    PubMed Central

    Reynolds, K A; Gerba, C P; Pepper, I L

    1996-01-01

    Rapid detection of infectious enteroviruses in environmental samples was made possible by utilizing an integrated cell culture-reverse transcriptase PCR approach. By this method, the presence of infectious enterovirus was confirmed within 24 h, compared with > or = 3 days by cell culture alone. The combined methodology eliminated typical problems normally associated with direct reverse transcriptase PCR by increasing the equivalent volume of environmental sample examined and reducing the effects of inhibitory compounds. PMID:8919804

  15. 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

  16. Detection of infectious laryngotracheitis virus by real-time PCR in naturally and experimentally infected chickens.

    PubMed

    Zhao, Yan; Kong, Congcong; Cui, Xianlan; Cui, Hongyu; Shi, Xingming; Zhang, Xiaomin; Hu, Shunlei; Hao, Lianwei; Wang, Yunfeng

    2013-01-01

    Infectious laryngotracheitis (ILT) is an acute, highly contagious upper-respiratory infectious disease of chickens. In this study, a real-time PCR method was developed for fast and accurate detection and quantitation of ILTV DNA of chickens experimentally infected with ILTV strain LJS09 and naturally infected chickens. The detection lower limit of the assay was 10 copies of DNA. There were no cross reactions with the DNA and RNA of infectious bursal disease virus, chicken anemia virus, reticuloendotheliosis virus, avian reovirus, Newcastle disease virus, and Marek's disease virus. The real-time PCR was reproducible as the coefficients of variation of reproducibility of the intra-assay and the inter-assay were less than 2%. The real-time PCR was used to detect the levels of the ILTV DNA in the tissues of specific pathogen free (SPF) chickens infected with ILTV at different times post infection. ILTV DNA was detected by real-time PCR in the heart, liver, spleen, lung, kidney, larynx, tongue, thymus, glandular stomach, duodenum, pancreatic gland, small intestine, large intestine, cecum, cecal tonsil, bursa of Fabricius, and brain of chickens in the infection group and the contact-exposure group. The sensitivity, specificity, and reproducibility of the ILTV real-time PCR assay revealed its suitability for detection and quantitation of ILTV in the samples from clinically and experimentally ILTV infected chickens. PMID:23840745

  17. Detection of Infectious Laryngotracheitis Virus by Real-Time PCR in Naturally and Experimentally Infected Chickens

    PubMed Central

    Zhao, Yan; Kong, Congcong; Cui, Xianlan; Cui, Hongyu; Shi, Xingming; Zhang, Xiaomin; Hu, Shunlei; Hao, Lianwei; Wang, Yunfeng

    2013-01-01

    Infectious laryngotracheitis (ILT) is an acute, highly contagious upper-respiratory infectious disease of chickens. In this study, a real-time PCR method was developed for fast and accurate detection and quantitation of ILTV DNA of chickens experimentally infected with ILTV strain LJS09 and naturally infected chickens. The detection lower limit of the assay was 10 copies of DNA. There were no cross reactions with the DNA and RNA of infectious bursal disease virus, chicken anemia virus, reticuloendotheliosis virus, avian reovirus, Newcastle disease virus, and Marek's disease virus. The real-time PCR was reproducible as the coefficients of variation of reproducibility of the intra-assay and the inter-assay were less than 2%. The real-time PCR was used to detect the levels of the ILTV DNA in the tissues of specific pathogen free (SPF) chickens infected with ILTV at different times post infection. ILTV DNA was detected by real-time PCR in the heart, liver, spleen, lung, kidney, larynx, tongue, thymus, glandular stomach, duodenum, pancreatic gland, small intestine, large intestine, cecum, cecal tonsil, bursa of Fabricius, and brain of chickens in the infection group and the contact-exposure group. The sensitivity, specificity, and reproducibility of the ILTV real-time PCR assay revealed its suitability for detection and quantitation of ILTV in the samples from clinically and experimentally ILTV infected chickens. PMID:23840745

  18. Application of multiplex PCR for Rapid and sensitive detection of human papillomaviruses in cervical cancer

    PubMed Central

    Zandnia, Fateme; Doosti, Abbas; Mokhtari-Farsani, Abbas; Kardi, Mohammad Taghi; Movafagh, Abolfazl

    2016-01-01

    Objectives: Reffering to an increase in cervical cancer in the recent years, rapid, sensitive and economical detection of human papillomaviruses (HPVs) as causative agents of cervical cancer is important. The traditional methods for the detection of HPVs in cervical cancer, such as pap smear, suffer from limitation and PCR has a potential to overcome the limitaitons. The purpose of present research work was to identify the five important strains of HPV (16, 18, 31, 33 and 45) simultaneously by Multiplex PCR application. Methods: Study was done on 100 cervical lesions of women. DNA was extracted from specimens by a genomic DNA purification kit. A 5-plex PCR was developed for the simultaneous detection of major HPV. Five pair of new primers was designed for detection of HPV 16, 18, 31, 33 and 45 by Multiplex PCR. Results: Among the 100 evaluated samples, 82 were found positive to HPVs. In the meantime the highest rate of infection was for HPV 16. Also 30 of HPV positive samples had infections with two or more HPV types. Conclusion: Multiplex PCR assay used in present study can provide a rapid, sensitive and economical method for detection of viral infections and is applicable to small volumes of vaginal samples. PMID:27182258

  19. 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

  20. Development of a nanoparticle-assisted PCR assay for detection of porcine epidemic diarrhea virus.

    PubMed

    Yuan, Wanzhe; Li, Yanan; Li, Peng; Song, Qinye; Li, Limin; Sun, Jiguo

    2015-08-01

    Porcine epidemic diarrhea virus (PEDV) is an important pig pathogen that can cause vomiting, diarrhea, and dehydration, leading to serious damage to the swine industry worldwide. In this study, a nanoparticle-assisted polymerase chain reaction (nanoPCR) assay targeting the N gene of PEDV was developed and the sensitivity and specificity were investigated. Under the optimized conditions for detection of PEDV RNA, the nanoPCR assay was 100-fold more sensitive than a conventional RT-PCR assay. The lower detection limit of the nanoPCR assay was 2.7 × 10(-6) ng/μL of PEDV RNA and no cross-reaction was observed with other viruses. This is the first report to demonstrate the application of a nanoPCR assay for the detection of PEDV. The sensitive and specific nanoPCR assay developed in this study can be applied widely in clinical diagnosis and field surveillance of PEDV-infection. PMID:25887451

  1. Organic Substances Interfere with Reverse Transcription-Quantitative PCR-Based Virus Detection in Water Samples

    PubMed Central

    Katayama, Hiroyuki; Furumai, Hiroaki

    2014-01-01

    Reverse transcription (RT)-PCR-based virus detection from water samples is occasionally hampered by organic substances that are coconcentrated during virus concentration procedures. To characterize these organic substances, samples containing commercially available humic acid, which is known to inhibit RT-PCR, and river water samples were subjected to adsorption-elution-based virus concentration using an electronegative membrane. In this study, the samples before, during, and after the concentration were analyzed in terms of organic properties and virus detection efficiencies. Two out of the three humic acid solutions resulted in RT-quantitative PCR (qPCR) inhibition that caused >3-log10-unit underestimation of spiked poliovirus. Over 60% of the organics contained in the two solutions were recovered in the concentrate, while over 60% of the organics in the uninhibited solution were lost during the concentration process. River water concentrates also caused inhibition of RT-qPCR. Organic concentrations in the river water samples increased by 2.3 to 3.9 times after the virus concentration procedure. The inhibitory samples contained organic fractions in the 10- to 100-kDa size range, which are suspected to be RT-PCR inhibitors. According to excitation-emission matrices, humic acid-like and protein-like fractions were also recovered from river water concentrates, but these fractions did not seem to affect virus detection. Our findings reveal that detailed organic analyses are effective in characterizing inhibitory substances. PMID:25527552

  2. Comparison of dermatophyte PCR kit with conventional methods for detection of dermatophytes in skin specimens.

    PubMed

    Kondori, Nahid; Tehrani, Parisa Afshari; Strömbeck, Louise; Faergemann, Jan

    2013-10-01

    The laboratory diagnosis of dermatophytosis is usually based on direct microscopic examination and culturing of clinical specimens. A commercial polymerase chain reaction kit (Dermatophyte PCR) has had favorable results when used for detection of dermatophytes and identification of Trichophyton rubrum in nail specimens. This study investigated the efficacy of the Dermatophyte PCR kit for detecting dermatophytosis in 191 hair or skin specimens from patients with suspected dermatophytosis. PCR was positive for 37 % of samples, whereas 31 and 39 % of the specimens were positive by culturing and direct microscopy, respectively. The sensitivity, specificity, positive predictive value, and negative predictive value for PCR analysis were 83, 84, 71, and 91 %, respectively. The sensitivity of the PCR test was higher in specimens obtained from skin (88 %) than in those obtained from hair (58 %), while the specificity remained almost the same (84 and 86 % for skin and hair, respectively). Our results show that the Dermatophyte PCR kit is a promising diagnostic tool for detection of dermatophytosis in skin samples, providing clinicians with a rapid diagnosis. PMID:23948965

  3. Evaluation of the Broad-Range PCR-Electrospray Ionization Mass Spectrometry (PCR/ESI-MS) System and Virus Microarrays for Virus Detection

    PubMed Central

    Taliaferro, Lanyn P.; Galvin, Teresa A.; Ma, Hailun; Shaheduzzaman, Syed; Williams, Dhanya K.; Glasner, Dustin R.; Khan, Arifa S.

    2014-01-01

    Advanced nucleic acid-based technologies are powerful research tools for novel virus discovery but need to be standardized for broader applications such as virus detection in biological products and clinical samples. We have used well-characterized retrovirus stocks to evaluate the limit of detection (LOD) for broad-range PCR with electrospray ionization mass spectrometry (PCR/ESI-MS or PLEX-ID), RT-PCR assays, and virus microarrays. The results indicated that in the absence of background cellular nucleic acids, PLEX-ID and RT-PCR had a similar LOD for xenotropic murine retrovirus-related virus (XMRV; 3.12 particles per µL) whereas sensitivity of virus detection was 10-fold greater using virus microarrays. When virus was spiked into a background of cellular nucleic acids, the LOD using PLEX-ID remained the same, whereas virus detection by RT-PCR was 10-fold less sensitive, and no virus could be detected by microarrays. Expected endogenous retrovirus (ERV) sequences were detected in cell lines tested and known species-specific viral sequences were detected in bovine serum and porcine trypsin. A follow-up strategy was developed using PCR amplification, nucleotide sequencing, and bioinformatics to demonstrate that an RD114-like retrovirus sequence that was detected by PLEX-ID in canine cell lines (Madin-Darby canine kidney (MDCK) and Cf2Th canine thymus) was due to defective, endogenous gammaretrovirus-related sequences. PMID:24777034

  4. Development of PCR and TaqMan PCR Assays to Detect Pseudomonas coronafaciens, a Causal Agent of Halo Blight of Oats

    PubMed Central

    An, Ji-Hye; Noh, Young-Hee; Kim, Yong-Eon; Lee, Hyok-In; Cha, Jae-Soon

    2015-01-01

    Pseudomonas coronafaciens causes halo blight on oats and is a plant quarantine bacterium in many countries, including the Republic of Korea. Using of the certificated seed is important for control of the disease. Since effective detection method of P. coronafaciens is not available yet, PCR and TaqMan PCR assays for specific detection of P. coronafaciens were developed in this study. PCR primers were designed from the draft genome sequence of P. coronafaciens LMG 5060 which was obtained by the next-generation sequencing in this study. The PCR primer set Pc-12-F/Pc-12-R specifically amplified 498 bp from the 13 strains of P. coronafaciens isolated in the seven different countries (Canada, Japan, United Kingdom, Zimbabwe, Kenya, Germany, and New Zealand) and the nested primer set Pc-12-ne-F/Pc-12-ne-R specifically amplified 298 bp from those strains. The target-size PCR product was not amplified from the non-target bacteria with the PCR and nested primer sets. TaqMan PCR with Pc-12-ne-F/Pc-12-ne-R and a TaqMan probe, Pc-taqman, which were designed inside of the nested PCR amplicon, generated Ct values which in a dose-dependent manner to the amount of the target DNA and the Ct values of all the P. coronafaciens strains were above the threshold Ct value for positive detection. The TaqMan PCR generated positive Ct values from the seed extracts of the artificially inoculated oat seeds above 10 cfu/ml inoculation level. PCR and TaqMan PCR assays developed in this study will be useful tools to detect and identify the plant quarantine pathogen, P. coronafaciens. PMID:25774107

  5. A new trilocus sequence-based multiplex-PCR to detect major Acinetobacter baumannii clones.

    PubMed

    Martins, Natacha; Picão, Renata Cristina; Cerqueira-Alves, Morgana; Uehara, Aline; Barbosa, Lívia Carvalho; Riley, Lee W; Moreira, Beatriz Meurer

    2016-08-01

    A collection of 163 Acinetobacter baumannii isolates detected in a large Brazilian hospital, was potentially related with the dissemination of four clonal complexes (CC): 113/79, 103/15, 109/1 and 110/25, defined by University of Oxford/Institut Pasteur multilocus sequence typing (MLST) schemes. The urge of a simple multiplex-PCR scheme to specify these clones has motivated the present study. The established trilocus sequence-based typing (3LST, for ompA, csuE and blaOXA-51-like genes) multiplex-PCR rapidly identifies international clones I (CC109/1), II (CC118/2) and III (CC187/3). Thus, the system detects only one (CC109/1) out of four main CC in Brazil. We aimed to develop an alternative multiplex-PCR scheme to detect these clones, known to be present additionally in Africa, Asia, Europe, USA and South America. MLST, performed in the present study to complement typing our whole collection of isolates, confirmed that all isolates belonged to the same four CC detected previously. When typed by 3LST-based multiplex-PCR, only 12% of the 163 isolates were classified into groups. By comparative sequence analysis of ompA, csuE and blaOXA-51-like genes, a set of eight primers was designed for an alternative multiplex-PCR to distinguish the five CC 113/79, 103/15, 109/1, 110/25 and 118/2. Study isolates and one CC118/2 isolate were blind-tested with the new alternative PCR scheme; all were correctly clustered in groups of the corresponding CC. The new multiplex-PCR, with the advantage of fitting in a single reaction, detects five leading A. baumannii clones and could help preventing the spread in healthcare settings. PMID:27125687

  6. Screening PCR Versus Sanger Sequencing: Detection of CALR Mutations in Patients With Thrombocytosis

    PubMed Central

    Jeong, Ji Hun; Lee, Hwan Tae; Seo, Ja Young; Seo, Yiel Hea; Kim, Kyung Hee; Kim, Moon Jin; Lee, Jae Hoon; Park, Jinny; Hong, Jun Shik

    2016-01-01

    Background Mutations in calreticulin (CALR) have been reported to be key markers in the molecular diagnosis of myeloid proliferative neoplasms. In most previous reports, CALR mutations were analyzed by using Sanger sequencing. Here, we report a new, rapid, and convenient system for screening CALR mutations without sequencing. Methods Eighty-three bone marrow samples were obtained from 81 patients with thrombocytosis. PCR primers were designed to detect wild-type CALR (product: 357 bp) and CALR with type 1 (product: 302 bp) and type 2 mutations (product: 272 bp) in one reaction. The results were confirmed by Sanger sequencing and compared with results from fragment analysis. Results The minimum detection limit of the screening PCR was 10 ng for type 1, 1 ng for type 2, and 0.1 ng for cases with both mutations. CALR type 1 and type 2 mutants were detected with screening PCR with a maximal analytical sensitivity of 3.2% and <0.8%, respectively. The screening PCR detected 94.1% (16/17) of mutation cases and showed concordant results with sequencing in the cases of type 1 and type 2 mutations. Sanger sequencing identified one novel mutation (c.1123_1132delinsTGC). Compared with sequencing, the screening PCR showed 94.1% sensitivity, 100.0% specificity, 100.0% positive predictive value, and 98.5% negative predictive value. Compared with fragment analysis, the screening PCR presented 88.9% sensitivity and 100.0% specificity. Conclusions This screening PCR is a rapid, sensitive, and cost-effective method for the detection of major CALR mutations. PMID:27139600

  7. 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. PMID:26991290

  8. PCR detection of bacterial genes provides evidence of death by drowning.

    PubMed

    Suto, Miwako; Kato, Naho; Abe, Sumiko; Nakamura, Masahide; Tsuchiya, Reo; Hiraiwa, Kouichi

    2009-04-01

    We have developed a sensitive and specific PCR method for detecting plankton DNA in cases of death by drowning. However, this PCR method could not be used for cases of drowning in water containing no plankton. Bacteria species are normally localized in the throat and trachea and they may invade into blood through the respiratory tract in people who have drowned as well as species localized in water. The aim of this study was to establish a novel and expedient PCR method for detecting bacterial genes in samples from drowning cases. We designed primer pairs for Streptococcus salivarius (SL1) and Streptococcus sanguinis (SN1), which are common species in the throat, and for Aeromonas hydrophila (AH1), which has been found in various water samples. With SL1, SN1, and AH1, we detected 10, 0.1, and 1 pg of target DNA, respectively. Among 19 drowned cases within 3 days postmortem, SL-DNA was detected in all of the blood samples from hearts with SL1 and AH-DNA was detected in several samples with AH1. In a case of drowning in a bathtub, use of the conventional acid digestion method for diatom analyses and the PCR method for identifying plankton DNA revealed no plankton, but our PCR method for detecting bacterial DNA showed a positive result for SL-DNA in a blood sample from the heart. In conclusion, our novel PCR method is highly specific and sensitive for detecting bacterial DNA and is useful for cases of death by drowning in water containing no plankton. PMID:19264526

  9. Low-Level Detection and Quantitation of Cellular HIV-1 DNA and 2-LTR Circles Using Droplet Digital PCR

    PubMed Central

    Henrich, Timothy J.; Gallien, Sebastien; Li, Jonathan Z.; Pereyra, Florencia; Kuritzkes, Daniel R.

    2012-01-01

    Droplet digital PCR (ddPCR) is an emerging nucleic acid detection method that provides absolute quantitations of target sequences without relying on the use of standard curves. The ability of ddPCR to detect and quantitate total HIV-1 DNA and 2-LTR circles from a panel of patients on and off antiviral therapy was evaluated compared to established real-time (RT)-PCR methods. To calculate the dynamic range of ddPCR for HIV-1 DNA and 2-LTR circles, serial dilutions of DNA amplicons or episomes were determined by ddPCR as well as with RT-PCR. HIV-1 DNA from 3 viremic patients and 4 patients on suppressive antiretroviral therapy, and 2-LTR circles from 3 patients with low-level viremia was also quantitated. Copy numbers determined by ddPCR of serial dilutions of HIV-1 or human CCR5 DNA amplicon standards were comparable to nominal input copy number. The sensitivity of ddPCR to detect HIV-1 or CCR5 DNA was similar to that of RT-PCR. Low levels of 2-LTR circles were detected in samples from all 3 patients by both ddPCR and RT-PCR. ddPCR is a promising novel technology for the study of HIV-1 reservoirs and persistence, but further optimization of this novel technology would enhance the detection of very low-level viral genetic targets. PMID:22974526

  10. Low-level detection and quantitation of cellular HIV-1 DNA and 2-LTR circles using droplet digital PCR.

    PubMed

    Henrich, Timothy J; Gallien, Sebastien; Li, Jonathan Z; Pereyra, Florencia; Kuritzkes, Daniel R

    2012-12-01

    Droplet digital PCR (ddPCR) is an emerging nucleic acid detection method that provides absolute quantitations of target sequences without relying on the use of standard curves. The ability of ddPCR to detect and quantitate total HIV-1 DNA and 2-LTR circles from a panel of patients on and off antiviral therapy was evaluated compared to established real-time (RT)-PCR methods. To calculate the dynamic range of ddPCR for HIV-1 DNA and 2-LTR circles, serial dilutions of DNA amplicons or episomes were determined by ddPCR as well as with RT-PCR. HIV-1 DNA from 3 viremic patients and 4 patients on suppressive antiretroviral therapy, and 2-LTR circles from 3 patients with low-level viremia were also quantitated. Copy numbers determined by ddPCR of serial dilutions of HIV-1 or human CCR5 DNA amplicon standards were comparable to nominal input copy number. The sensitivity of ddPCR to detect HIV-1 or CCR5 DNA was similar to that of RT-PCR. Low levels of 2-LTR circles were detected in samples from all 3 patients by both ddPCR and RT-PCR. ddPCR is a promising novel technology for the study of HIV-1 reservoirs and persistence, but further optimization of this novel technology would enhance the detection of very low-level viral genetic targets. PMID:22974526

  11. Detection of Histoplasma capsulatum from clinical specimens by cycling probe-based real-time PCR and nested real-time PCR.

    PubMed

    Muraosa, Yasunori; Toyotome, Takahito; Yahiro, Maki; Watanabe, Akira; Shikanai-Yasuda, Maria Aparecida; Kamei, Katsuhiko

    2016-05-01

    We developed new cycling probe-based real-time PCR and nested real-time PCR assays for the detection of Histoplasma capsulatum that were designed to detect the gene encoding N-acetylated α-linked acidic dipeptidase (NAALADase), which we previously identified as an H. capsulatum antigen reacting with sera from patients with histoplasmosis. Both assays specifically detected the DNAs of all H. capsulatum strains but not those of other fungi or human DNA. The limited of detection (LOD) of the real-time PCR assay was 10 DNA copies when using 10-fold serial dilutions of the standard plasmid DNA and 50 DNA copies when using human serum spiked with standard plasmid DNA. The nested real-time PCR improved the LOD to 5 DNA copies when using human serum spiked with standard plasmid DNA, which represents a 10-fold higher than that observed with the real-time PCR assay. To assess the ability of the two assays to diagnose histoplasmosis, we analyzed a small number of clinical specimens collected from five patients with histoplasmosis, such as sera (n = 4), formalin-fixed paraffin-embedded (FFPE) tissue (n = 4), and bronchoalveolar lavage fluid (BALF) (n = 1). Although clinical sensitivity of the real-time PCR assay was insufficiently sensitive (33%), the nested real-time PCR assay increased the clinical sensitivity (77%), suggesting it has a potential to be a useful method for detecting H. capsulatum DNA in clinical specimens. PMID:26705837

  12. Detection of acute toxoplasmosis in pigs using loop-mediated isothermal amplification and quantitative PCR.

    PubMed

    Wang, Yanhua; Wang, Guangxiang; Zhang, Delin; Yin, Hong; Wang, Meng

    2013-10-01

    A loop-mediated isothermal amplification (LAMP) assay allows rapid diagnosis of Toxoplasma gondii infection. In the present study, the LAMP assay was evaluated using blood from both naturally and experimentally infected pigs. The sensitivity of the LAMP assay was compared with that of Q-PCR. Both assays detected T. gondii in the blood of experimentally infected pigs, with 100% agreement. In infected blood samples, the parasite was detected as early as 2 days post-infection and reached a peak in 3-5 days. In 216 field serum samples, the detection rates of LAMP and Q-PCR assays were 6.9% and 7.8%, respectively. This result indicates that the sensitivity of the LAMP assay was slightly lower than that of the Q-PCR assay. However, the LAMP may be an attractive diagnostic method in conditions where sophisticated and expensive equipment is unavailable. This assay could be a powerful supplement to current diagnostic methods. PMID:24327785

  13. Multiplex PCR for the concurrent detection and differentiation of Salmonella spp., Salmonella Typhi and Salmonella Typhimurium

    PubMed Central

    Pui, Chai Fung; Wong, Woan Chwen; Chai, Lay Ching; Lee, Hai Yen; Noorlis, Ahmad; Zainazor, Tuan Chilek Tuan; Tang, John Yew Huat; Ghazali, Farinazleen Mohamad; Cheah, Yoke Kqueen; Nakaguchi, Yoshitsugu; Nishibuchi, Mitsuaki; Radu, Son

    2011-01-01

    Salmonellosis outbreaks involving typhoid fever and human gastroenteritis are important diseases in tropical countries where hygienic conditions are often not maintained. A rapid and sensitive method to detect Salmonella spp., Salmonella Typhi and Salmonella Typhimurium is needed to improve control and surveillance of typhoid fever and Salmonella gastroenteritis. Our objective was the concurrent detection and differentiation of these food-borne pathogens using a multiplex PCR. We therefore designed and optimized a multiplex PCR using three specific PCR primer pairs for the simultaneous detection of these pathogens. The concentration of each of the primer pairs, magnesium chloride concentration, and primer annealing temperature were optimized before verification of the specificity of the primer pairs. The target genes produced amplicons at 429 bp, 300 bp and 620 bp which were shown to be 100% specific to each target bacterium, Salmonella spp., Salmonella Typhi and Salmonella Typhimurium, respectively. PMID:22028607

  14. NINA-LAMP compared to microscopy, RDT, and nested PCR for the detection of imported malaria.

    PubMed

    Mohon, Abu Naser; Lee, Lydia Da-Yeong; Bayih, Abebe Genetu; Folefoc, Asongna; Guelig, Dylan; Burton, Robert A; LaBarre, Paul; Chan, Wilson; Meatherall, Bonnie; Pillai, Dylan R

    2016-06-01

    Microscopy and field adaptable rapid diagnostic tests (RDTs) are not sensitive and specific in certain conditions such as poor training of microscopists, lack of electricity, or lower sensitivity in the detection of non-falciparum malaria. More sensitive point-of-care testing (POCT) would reduce delays in diagnosis and initiation of therapy. In the current study, we have evaluated the efficacy of noninstrumented nucleic acid amplification (NINA) coupled with loop-mediated isothermal amplification (LAMP) for detection of traveler's malaria (n=140) in comparison with microscopy, nested PCR, and the only Food and Drug Administration-approved rapid diagnostic test. NINA-LAMP was 100% sensitive and 98.6% specific when compared to nested PCR. For non-falciparum detection, NINA-LAMP sensitivity was 100% sensitive compared to nested PCR, whereas RDT sensitivity was 71%. LAMP is highly sensitive and specific for symptomatic malaria diagnosis regardless of species. PMID:27017271

  15. Validation of a quantitative PCR assay for detection and quantification of 'Candidatus Xenohaliotis californiensis'.

    PubMed

    Friedman, Carolyn S; Wight, Nate; Crosson, Lisa M; White, Samuel J; Strenge, Robyn M

    2014-04-01

    Withering syndrome (WS), a serious disease affecting abalone Haliotis spp., is caused by infection from an intracellular Rickettsia-like organism (WS-RLO). Diagnosis of the disease currently relies on a combination of histological examination and molecular methods (in situ hybridization, standard PCR, and sequence analysis). However, these techniques only provide a semi-quantitative assessment of bacterial load. We created a real-time quantitative PCR (qPCR) assay to specifically identify and enumerate bacterial loads of WS-RLO in abalone tissue, fecal, and seawater samples based on 16S rDNA gene copy numbers. The qPCR assay designed to detect DNA of the WS-RLO was validated according to standards set by the World Organisation for Animal Health. Standard curves derived from purified plasmid dilutions were linear across 7 logs of concentration, and efficiencies ranged from 90.2 to 97.4%. The limit of detection was 3 gene copies per reaction. Diagnostic sensitivity was 100% and specificity was 99.8%. The qPCR assay was robust, as evidenced by its high level of repeatability and reproducibility. This study has shown for the first time that WS-RLO DNA can be detected and quantified in abalone tissue, fecal, and seawater samples. The ability to detect and quantify RLO gene copies in a variety of materials will enable us to better understand transmission dynamics in both farmed and natural environments. PMID:24695238

  16. A point-of-care PCR test for HIV-1 detection in resource-limited settings.

    PubMed

    Jangam, Sujit R; Agarwal, Abhishek K; Sur, Kunal; Kelso, David M

    2013-04-15

    A low-cost, fully integrated sample-to-answer, quantitative PCR (qPCR) system that can be used for detection of HIV-1 proviral DNA in infants at the point-of-care in resource-limited settings has been developed and tested. The system is based on a novel DNA extraction method, which uses a glass fiber membrane, a disposable assay card that includes on-board reagent storage, provisions for thermal cycling and fluorescence detection, and a battery-operated portable analyzer. The system is capable of automated PCR mix assembly using a novel reagent delivery system and performing qPCR. HIV-1 and internal control targets are detected using two spectrally separated fluorophores, FAM and Quasar 670. In this report, a proof-of-concept of the platform is demonstrated. Initial results with whole blood demonstrate that the test is capable of detecting HIV-1 in blood samples containing greater than 5000 copies of HIV-1. In resource-limited settings, a point-of-care HIV-1 qPCR test would greatly increase the number of test results that reach the infants caregivers, allowing them to pursue anti-retroviral therapy. PMID:23202333

  17. Multiplex PCR for Detection of Botulinum Neurotoxin-Producing Clostridia in Clinical, Food, and Environmental Samples▿

    PubMed Central

    De Medici, Dario; Anniballi, Fabrizio; Wyatt, Gary M.; Lindström, Miia; Messelhäußer, Ute; Aldus, Clare F.; Delibato, Elisabetta; Korkeala, Hannu; Peck, Michael W.; Fenicia, Lucia

    2009-01-01

    Botulinum neurotoxin (BoNT), the most toxic substance known, is produced by the spore-forming bacterium Clostridium botulinum and, in rare cases, also by some strains of Clostridium butyricum and Clostridium baratii. The standard procedure for definitive detection of BoNT-producing clostridia is a culture method combined with neurotoxin detection using a standard mouse bioassay (SMB). The SMB is highly sensitive and specific, but it is expensive and time-consuming and there are ethical concerns due to use of laboratory animals. PCR provides a rapid alternative for initial screening for BoNT-producing clostridia. In this study, a previously described multiplex PCR assay was modified to detect all type A, B, E, and F neurotoxin genes in isolated strains and in clinical, food, environmental samples. This assay includes an internal amplification control. The effectiveness of the multiplex PCR method for detecting clostridia possessing type A, B, E, and F neurotoxin genes was evaluated by direct comparison with the SMB. This method showed 100% inclusivity and 100% exclusivity when 182 BoNT-producing clostridia and 21 other bacterial strains were used. The relative accuracy of the multiplex PCR and SMB was evaluated using 532 clinical, food, and environmental samples and was estimated to be 99.2%. The multiplex PCR was also used to investigate 110 freshly collected food and environmental samples, and 4 of the 110 samples (3.6%) were positive for BoNT-encoding genes. PMID:19684163

  18. Multiplex PCR for detection of botulinum neurotoxin-producing clostridia in clinical, food, and environmental samples.

    PubMed

    De Medici, Dario; Anniballi, Fabrizio; Wyatt, Gary M; Lindström, Miia; Messelhäusser, Ute; Aldus, Clare F; Delibato, Elisabetta; Korkeala, Hannu; Peck, Michael W; Fenicia, Lucia

    2009-10-01

    Botulinum neurotoxin (BoNT), the most toxic substance known, is produced by the spore-forming bacterium Clostridium botulinum and, in rare cases, also by some strains of Clostridium butyricum and Clostridium baratii. The standard procedure for definitive detection of BoNT-producing clostridia is a culture method combined with neurotoxin detection using a standard mouse bioassay (SMB). The SMB is highly sensitive and specific, but it is expensive and time-consuming and there are ethical concerns due to use of laboratory animals. PCR provides a rapid alternative for initial screening for BoNT-producing clostridia. In this study, a previously described multiplex PCR assay was modified to detect all type A, B, E, and F neurotoxin genes in isolated strains and in clinical, food, environmental samples. This assay includes an internal amplification control. The effectiveness of the multiplex PCR method for detecting clostridia possessing type A, B, E, and F neurotoxin genes was evaluated by direct comparison with the SMB. This method showed 100% inclusivity and 100% exclusivity when 182 BoNT-producing clostridia and 21 other bacterial strains were used. The relative accuracy of the multiplex PCR and SMB was evaluated using 532 clinical, food, and environmental samples and was estimated to be 99.2%. The multiplex PCR was also used to investigate 110 freshly collected food and environmental samples, and 4 of the 110 samples (3.6%) were positive for BoNT-encoding genes. PMID:19684163

  19. 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

  20. Diagnosis of bacterial kidney disease by detection of Renibacterium salmoninarum by real-time PCR.

    PubMed

    Jansson, E; Lindberg, L; Säker, E; Aspán, A

    2008-10-01

    Bacterial kidney disease (BKD), caused by Renibacterium salmoninarum (Rs), is a serious threat to salmon in aquaculture as well as to wild populations. We have developed a real-time polymerase chain reaction (PCR) for detection of Rs in kidney samples. The PCR is based on detection of unique parts of the 16S rRNA gene of Rs and DNA equivalent to 1-10 Rs genomes was detected per reaction. No cross-reactivity with other fish pathogenic or related bacteria could be demonstrated. Analysis of individual kidney samples collected from BKD classified populations identified 39.9% of the fish as positive by real-time PCR compared with 28.0% by polyclonal enzyme-linked immunosorbent assay (ELISA). The real-time PCR assay was found to be well suited for complementary use with ELISA for diagnosis of BKD, with the ability to detect clinical as well as covert Rs infections. The infection level determined by the polyclonal ELISA and by real-time PCR was significantly correlated. PMID:18681904

  1. PCR detection of malaria parasites in desiccated Anopheles mosquitoes is uninhibited by storage time and temperature

    PubMed Central

    2012-01-01

    Background Reliable methods to preserve mosquito vectors for malaria studies are necessary for detecting Plasmodium parasites. In field settings, however, maintaining a cold chain of storage from the time of collection until laboratory processing, or accessing other reliable means of sample preservation is often logistically impractical or cost prohibitive. As the Plasmodium infection rate of Anopheles mosquitoes is a central component of the entomological inoculation rate and other indicators of transmission intensity, storage conditions that affect pathogen detection may bias malaria surveillance indicators. This study investigated the effect of storage time and temperature on the ability to detect Plasmodium parasites in desiccated Anopheles mosquitoes by real-time polymerase chain reaction (PCR). Methods Laboratory-infected Anopheles stephensi mosquitoes were chloroform-killed and stored over desiccant for 0, 1, 3, and 6 months while being held at four different temperatures: 28, 37, -20 and -80°C. The detection of Plasmodium DNA was evaluated by real-time PCR amplification of a 111 base pair region of block 4 of the merozoite surface protein. Results Varying the storage time and temperature of desiccated mosquitoes did not impact the sensitivity of parasite detection. A two-way factorial analysis of variance suggested that storage time and temperature were not associated with a loss in the ability to detect parasites. Storage of samples at 28°C resulted in a significant increase in the ability to detect parasite DNA, though no other positive associations were observed between the experimental storage treatments and PCR amplification. Conclusions Cold chain maintenance of desiccated mosquito samples is not necessary for real-time PCR detection of parasite DNA. Though field-collected mosquitoes may be subjected to variable conditions prior to molecular processing, the storage of samples over an inexpensive and logistically accessible desiccant will likely

  2. Rapid detection of Ophiostoma piceae and O. quercus in stained wood by PCR.

    PubMed

    Kim, S H; Uzunovic, A; Breuil, C

    1999-01-01

    A rapid, sensitive, and simple method was developed to detect the sapstain fungi Ophiostoma piceae and O. quercus in stained wood. By using microwave heating for DNA extraction and PCR with internal transcribed spacer-derived-specific primers, detection was feasible within 4 h, even with DNA obtained from a single synnema. This method can easily be extended for the detection of other wood-inhabiting fungi. PMID:9872792

  3. Quantitative real-time PCR detection of Zika virus and evaluation with field-caught Mosquitoes

    PubMed Central

    2013-01-01

    Background Zika virus (ZIKV), a mosquito borne flavivirus is a pathogen affecting humans in Asia and Africa. ZIKV infection diagnosis relies on serology–which is challenging due to cross-reactions with other flaviviruses and/or absence or low titer of IgM and IgG antibodies at early phase of infection- virus isolation, which is labor intensive, time consuming and requires appropriate containment. Therefore, real-time RT-PCR (rRT-PCR) is an appealing option as a rapid, sensitive and specific method for detection of ZIKV in the early stage of infection. So far, only one rRT-PCR assay has been described in the context of the outbreak in Micronesia in 2007. In this study, we described a one step rRT-PCR for ZIKV which can detect a wider genetic diversity of ZIKV isolates from Asia and Africa. Results The NS5 protein coding regions of African ZIKV isolates were sequenced and aligned with representative flaviviruses sequences from GenBank to design primers and probe from conserved regions. The analytical sensitivity of the assay was evaluated to be 32 genome-equivalents and 0.05 plaque forming unit (pfu). The assay was shown to detect 37 ZIKV isolates covering a wide geographic in Africa and Asia over 36 years but none of the 31 other flaviviruses tested showing high analytical specificity. The rRT-PCR could be performed in less than 3 hours. This method was used successfully to detect ZIKV strains from field-caught mosquitoes. Conclusion We have developed a rapid, sensitive and specific rRT – PCR for detection of ZIKV. This assay is a useful tool for detection of ZIKV infection in regions where a number of other clinically indistinguishable arboviruses like dengue or chikungunya co-circulate. Further studies are needed to validate this assay in clinical positive samples collected during acute ZIKV infection. PMID:24148652

  4. Validation of nested PCR and a selective biochemical method as alternatives for mycoplasma detection.

    PubMed

    Cheong, Kyung Ah; Agrawal, Santosh Rani; Lee, Ai-Young

    2011-04-01

    Direct culture is the most common way to reliably detect mycoplasma, but it is not practical for the qualitative control of cell therapeutics because of the elaborate culture medium, the prolonged incubation time, and the large sample volumes. Here, we chose two alternative methods using commercial detection kits, the PCR mycoplasma detection kit with nested PCR and the selective biochemical method, MycoAlert(®), and validated them with the direct culture method as a reference. We tested eight mycoplasma species and five validation parameters: specificity, detection limit, robustness, repeatability, and ruggedness, based on the regulatory guidelines in the US Pharmacopoeia. All experiments were performed using fibroblasts spiked with mycoplasma. Specificity tests for both methods included all mycoplasma species, except Mycoplasma pneumonia and M. genitalium for the nested PCR and Ureaplasma urealyticum for the MycoAlert(®) assay. Regarding the detection limit, the nested PCR proved to be as sensitive as the direct culture method and more sensitive than the MycoAlert(®) assay. The predicted median for probit = 0.9 was 54 (44-76) CFU/ml for M. hyorhinis and 16 (13-23) CFU/ml for M. hominis by the nested PCR, but 431 (346-593) CFU/ml and 105 (87-142) CFU/ml, respectively, with MycoAlert(®). Changes in the concentration of reagents, reagent lot, or individual analysts did not influence the results of the examined methods. The results of this study support nested PCR as a valuable alternative for mycoplasma detection. PMID:20806253

  5. Rapid PCR Detection of Mycoplasma hominis, Ureaplasma urealyticum, and Ureaplasma parvum

    PubMed Central

    Cunningham, Scott A.; Mandrekar, Jayawant N.; Rosenblatt, Jon E.; Patel, Robin

    2013-01-01

    Objective. We compared laboratory developed real-time PCR assays for detection of Mycoplasma hominis and for detection and differentiation of Ureaplasma urealyticum and parvum to culture using genitourinary specimens submitted for M. hominis and Ureaplasma culture. Methods. 283 genitourinary specimens received in the clinical bacteriology laboratory for M. hominis and Ureaplasma species culture were evaluated. Nucleic acids were extracted using the Total Nucleic Acid Kit on the MagNA Pure 2.0. 5 μL of the extracts were combined with 15 μL of each of the two master mixes. Assays were performed on the LightCycler 480 II system. Culture was performed using routine methods. Results.  M. hominis PCR detected 38/42 M. hominis culture-positive specimens, as well as 2 that were culture negative (sensitivity, 90.5%; specificity, 99.2%). Ureaplasma PCR detected 139/144 Ureaplasma culture-positive specimens, as well as 9 that were culture negative (sensitivity, 96.5%; specificity, 93.6%). Of the specimens that tested positive for Ureaplasma species, U. urealyticum alone was detected in 33, U. parvum alone in 109, and both in 6. Conclusion. The described PCR assays are rapid alternatives to culture for detection of M. hominis and Ureaplasma species, and, unlike culture, the Ureaplasma assay easily distinguishes U. urealyticum from parvum. PMID:26904723

  6. Sensitive detection of cancer cells using light-mediated apta-PCR.

    PubMed

    Civit, Laia; Pinto, Alessandro; Rodrigues-Correia, Alexandre; Heckel, Alexander; O'Sullivan, Ciara K; Mayer, Günter

    2016-03-15

    Apta-PCR is an ultrasensitive assay in which aptamers are exploited not only as biomolecular recognition elements, but also as reporter labels for amplification via real-time PCR. This methodology has been successfully applied to the detection of proteins, achieving limits of detection in the picomolar range. The introduction of caged aptamers that bear photo-labile groups, so called cages, at strategic positions so that their tertiary structure and thus their binding properties can be controlled by light, facilitates a more robust and attractive assay in terms of sample conservation and reusability. In this work, we report for the first time the use of caged aptamers for cell detection in an apta-PCR assay. Specifically, a sandwich format is used combining the capture of B-cells by an antibody with the specific detection of Burkitt's lymphoma cancer cells by a caged aptamer, acting as a reporter probe. Elution of the aptamer bound to the cancer cells is performed by light and the number of cells is then correlated with the amount of eluted caged aptamer using real-time PCR analysis. The reported technique shows an excellent sensitivity, achieving detection of as few as 77 cells, and due to the inherent robustness of the assay, this detection platform can be reused for further analyses, demonstrating potential applicability in proteomics and clinical diagnostics. PMID:26615953

  7. Evaluation of the use of PCR and reverse transcriptase PCR for detection of pathogenic bacteria in biosolids from anaerobic digestors and aerobic composters.

    PubMed

    Burtscher, Carola; Wuertz, Stefan

    2003-08-01

    A PCR-based method and a reverse transcriptase PCR (RT-PCR)-based method were developed for the detection of pathogenic bacteria in organic waste, using Salmonella spp., Listeria monocytogenes, Yersinia enterocolitica, and Staphylococcus aureus as model organisms. In seeded organic waste samples, detection limits of less than 10 cells per g of organic waste were achieved after one-step enrichment of bacteria, isolation, and purification of DNA or RNA before PCR or RT-PCR amplification. To test the reproducibility and reliability of the newly developed methods, 46 unseeded samples were collected from diverse aerobic (composting) facilities and anaerobic digestors and analyzed by both culture-based classical and newly developed PCR-based procedures. No false-positive but some false-negative results were generated by the PCR- or RT-PCR-based methods after one-step enrichment when compared to the classical detection methods. The results indicated that the level of activity of the tested bacteria in unseeded samples was very low compared to that of freshly inoculated cells, preventing samples from reaching the cell density required for PCR-based detection after one-step enrichment. However, for Salmonella spp., a distinct PCR product could be obtained for all 22 nonamended samples that tested positive for Salmonella spp. by the classical detection procedure when a selective two-step enrichment (20 h in peptone water at 37 degrees C and 24 h in Rappaport Vassiliadis medium at 43 degrees C) was performed prior to nucleic acid extraction and PCR. Hence, the classical procedure was shortened, since cell plating and further differentiation of isolated colonies can be omitted, substituted for by highly sensitive and reliable detection based on nucleic acid extraction and PCR. Similarly, 2 of the 22 samples in which Salmonella spp. were detected also tested positive for Listeria monocytogenes according to a two-step enrichment procedure followed by PCR, compared to 3 samples

  8. 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.

  9. Optimization of PMA-PCR Protocol for Viability Detection of Pathogens

    NASA Technical Reports Server (NTRS)

    Mikkelson, Brian J.; Lee, Christine M.; Ponce, Adrian

    2011-01-01

    This presented study demonstrates the need that PMA-PCR can be used to capture the loss of viability of a sample that is much more specific and time-efficient than alternative methods. This protocol is particularly useful in scenarios in which sterilization treatments may inactivate organisms but not degrade their DNA. The use of a PCR-based method of pathogen detection without first inactivating the DNA of nonviable cells will potentially lead to false positives. The loss of culturability, by heat-killing, did not prevent amplified PCR products, which supports the use of PMA to prevent amplification and differentiate between viable and dead cells. PMA was shown to inhibit the amplification of DNA by PCR in vegetative cells that had been heat-killed.

  10. Goose Hemorrhagic polyomavirus detection in geese using real-time PCR assay.

    PubMed

    Leon, Olivier; Corrand, Léni; Bich, Tran Ngoc; Le Minor, Odile; Lemaire, Mylène; Guérin, Jean-Luc

    2013-12-01

    Goose hemorrhagic polyomavirus (GHPV) is the viral agent of hemorrhagic nephritis enteritis of geese (HNEG), a lethal disease of goslings. Although death is the most common outcome, geese that recover from HNEG are persistently infected. Here, we present the development of real-time SYBR Green real-time PCR targeted to GHPV and its use to assess the prevalence of GHPV infection in French geese flocks. When compared with classical end-point PCR, real-time PCR revealed a much better sensitivity and equivalent specificity. Real-time PCR could, therefore, be considered a gold standard for the detection of GHPV. Results of field investigations evidenced a very high prevalence of GHPV infections in French geese, largely associated with healthy carriage. PMID:24597124

  11. Molecular detection of Puccinia horiana in Chrysanthemum x morifolium through conventional and real-time PCR.

    PubMed

    Alaei, Hossein; Baeyen, Steve; Maes, Martine; Höfte, Monica; Heungens, Kurt

    2009-02-01

    Puccinia horiana Henn. is a quarantine organism and one of the most important fungal pathogens of Chrysanthemum x morifolium cultivars grown for cut flower or potted plant production (florist's chrysanthemum) in several regions of the world. Highly specific primer pairs were identified for conventional, nested, and real-time PCR detection of P. horiana based on the specific and sensitive PCR amplification of selected regions in the internal transcribed spacers (ITS1 and ITS2) of the nuclear ribosomal DNA (rDNA). Using these different PCR versions, 10 pg, 10 fg, and 5 fg genomic DNA could be detected, respectively. When using cloned target DNA as template, the detection limits were 5000, 50, and 5 target copies, respectively. These detection limits were not affected by a background of chrysanthemum plant DNA. The DNA extraction method was optimized to maximize the recoverability of the pathogen from infected plant tissue. A CTAB extraction protocol or a selection of commercial DNA extraction methods allowed the use of 10 ng total (plant+pathogen) DNA without interference of PCR inhibitors. Due to the specificity of the primers, SYBR Green I technology enabled reliable real time PCR signal detection. However, an efficient TaqMan probe is available. The lowest proportion of infected plant material that could still be detected when mixed with healthy plant material was 0.001%. The real-time PCR assay could detect as few as eight pure P. horiana basidiospores, demonstrating the potential of the technique for aerial detection of the pathogen. The amount of P. horiana DNA in plant tissue was determined at various time points after basidiospore inoculation. Using the real-time PCR protocol, it was possible to detect the pathogen immediately after the inoculation period, even though the accumulation of pathogen DNA was most pronounced near the end of the latent period. The detection system proved to be accurate and sensitive and could help not only in pathogen diagnosis but

  12. Rapid-Viability PCR Method for Detection of Live, Virulent Bacillus anthracis in Environmental Samples ▿

    PubMed Central

    Létant, Sonia E.; Murphy, Gloria A.; Alfaro, Teneile M.; Avila, Julie R.; Kane, Staci R.; Raber, Ellen; Bunt, Thomas M.; Shah, Sanjiv R.

    2011-01-01

    In the event of a biothreat agent release, hundreds of samples would need to be rapidly processed to characterize the extent of contamination and determine the efficacy of remediation activities. Current biological agent identification and viability determination methods are both labor- and time-intensive such that turnaround time for confirmed results is typically several days. In order to alleviate this issue, automated, high-throughput sample processing methods were developed in which real-time PCR analysis is conducted on samples before and after incubation. The method, referred to as rapid-viability (RV)-PCR, uses the change in cycle threshold after incubation to detect the presence of live organisms. In this article, we report a novel RV-PCR method for detection of live, virulent Bacillus anthracis, in which the incubation time was reduced from 14 h to 9 h, bringing the total turnaround time for results below 15 h. The method incorporates a magnetic bead-based DNA extraction and purification step prior to PCR analysis, as well as specific real-time PCR assays for the B. anthracis chromosome and pXO1 and pXO2 plasmids. A single laboratory verification of the optimized method applied to the detection of virulent B. anthracis in environmental samples was conducted and showed a detection level of 10 to 99 CFU/sample with both manual and automated RV-PCR methods in the presence of various challenges. Experiments exploring the relationship between the incubation time and the limit of detection suggest that the method could be further shortened by an additional 2 to 3 h for relatively clean samples. PMID:21764960

  13. Development of droplet digital PCR for the detection of Babesia microti and Babesia duncani

    PubMed Central

    Wilson, Melisa; Glaser, Kathleen C.; Adams-Fish, Debra; Boley, Matthew; Mayda, Maria; Molestina, Robert E.

    2014-01-01

    Babesia spp. are obligate protozoan parasites of red blood cells. Transmission to humans occurs through bites from infected ticks or blood transfusion. Infections with B. microti account for the majority of the reported cases of human babesiosis in the USA. A lower incidence is caused by the more recently described species B. duncani. The current gold standard for detection of Babesia is microscopic examination of blood smears. Recent PCR-based assays, including real-time PCR, have been developed for B. microti. On the other hand, molecular assays that detect and distinguish between B. microti and B. duncani infections are lacking. Closely related species of Babesia can be differentiated due to sequence variation within the internal transcribed spacer (ITS) regions of nuclear ribosomal RNAs. In the present study, we targeted the ITS regions of B. microti and B. duncani to develop sensitive and species-specific droplet digital PCR (ddPCR) assays. The assays were shown to discriminate B. microti from B. duncani and resulted in limits of detection of ~10 gene copies. Moreover, ddPCR for these species were useful in DNA extracted from blood of experimentally infected hamsters, detecting infections of low parasitemia that were negative by microscopic examination. In summary, we have developed sensitive and specific quantitative ddPCR assays for the detection of B. microti and B. duncani in blood. Our methods could be used as sensitive approaches to monitor the progression of parasitemia in rodent models of infection as well as serve as suitable molecular tests in blood screening. PMID:25500215

  14. Development of droplet digital PCR for the detection of Babesia microti and Babesia duncani.

    PubMed

    Wilson, Melisa; Glaser, Kathleen C; Adams-Fish, Debra; Boley, Matthew; Mayda, Maria; Molestina, Robert E

    2015-02-01

    Babesia spp. are obligate protozoan parasites of red blood cells. Transmission to humans occurs through bites from infected ticks or blood transfusion. Infections with B. microti account for the majority of the reported cases of human babesiosis in the USA. A lower incidence is caused by the more recently described species B. duncani. The current gold standard for detection of Babesia is microscopic examination of blood smears. Recent PCR-based assays, including real-time PCR, have been developed for B. microti. On the other hand, molecular assays that detect and distinguish between B. microti and B. duncani infections are lacking. Closely related species of Babesia can be differentiated due to sequence variation within the internal transcribed spacer (ITS) regions of nuclear ribosomal RNAs. In the present study, we targeted the ITS regions of B. microti and B. duncani to develop sensitive and species-specific droplet digital PCR (ddPCR) assays. The assays were shown to discriminate B. microti from B. duncani and resulted in limits of detection of ~10 gene copies. Moreover, ddPCR for these species were useful in DNA extracted from blood of experimentally infected hamsters, detecting infections of low parasitemia that were negative by microscopic examination. In summary, we have developed sensitive and specific quantitative ddPCR assays for the detection of B. microti and B. duncani in blood. Our methods could be used as sensitive approaches to monitor the progression of parasitemia in rodent models of infection as well as serve as suitable molecular tests in blood screening. PMID:25500215

  15. Detection of West Nile virus in mosquitoes by RT-PCR.

    PubMed

    Hadfield, T L; Turell, M; Dempsey, M P; David, J; Park, E J

    2001-06-01

    A reverse transcriptase-polymerase chain reaction (RT-PCR) assay employing detection technology was developed to identify West Nile virus in experimentally infected mosquitoes. The specificity of the assay was evaluated with the following viruses: eastern equine encephalitis, Ilheus, West Nile and yellow fever viruses. The limits of detection were determined using West Nile viral RNA extracted from serial dilutions of virus culture in infected mosquitoes. Limit of detection was 5 PFU from extracted mosquitoes. We were able to detect the presence of one infected mosquito in a pool of 50 repeatedly. When the RT-PCR was used with coded samples of intrathoracically-infected and uninfected mosquitoes, the assay detected the virus in all infected mosquitoes. Analytic sensitivity and specificity were 100%. This assay offers an efficient and rapid method of identifying West Nile virus in infected mosquitoes or cell culture. PMID:11352595

  16. An electrochemiluminescence non-PCR method for the detection of genetically modified organisms

    NASA Astrophysics Data System (ADS)

    Liu, Jinfeng; Xing, Da; Zhu, Debin

    2006-09-01

    An electrochemiluminescence non-PCR method has been developed for the detection of genetically modified organisms (GMOs) in crops. Genomic DNA of GMOs was digested with two restriction endonucleases (FOK I and BsrD I), and hybridized with three Ru(bpy) 3 2+ (TBR)-labeled and one biotinylated probes. The hybridization products were captured onto streptavidin-coated paramagnetic beads, and detected by measuring the electrochemiluminescence (ECL) signal of the TBR label. Whether the tobaccos contain GM components was discriminated by detecting the ECL signal of CaMV35S promoter. The experiment results show that the detection limit for CaMV35S promoter is 100 fmol, and the GM components can be clearly identified in GM tobaccos. The ECL non-PCR method will provide a new means in GMOs detection due to its safety, simplicity and high efficiency.

  17. Detection of mandarin in orange juice by single-nucleotide polymorphism qPCR assay.

    PubMed

    Aldeguer, Miriam; López-Andreo, María; Gabaldón, José A; Puyet, Antonio

    2014-02-15

    A dual-probe real time PCR (qPCR) DNA-based analysis was devised for the identification of mandarin in orange juice. A single nucleotide polymorphism at the trnL-trnF intergenic region of the chloroplast chromosome was confirmed in nine orange (Citrus sinensis) and thirteen commercial varieties of mandarin, including Citrus reticulata and Citrus unshiu species and a mandarin × tangelo hybrid. Two short minor-groove binding fluorescent probes targeting the polymorphic sequence were used in the dual-probe qPCR, which allowed the detection of both species in single-tube reactions. The similarity of PCR efficiencies allowed a simple estimation of the ratio mandarin/orange in the juice samples, which correlated to the measured difference of threshold cycle values for both probes. The limit of detection of the assay was 5% of mandarin in orange juice, both when the juice was freshly prepared (not from concentrate) or reconstituted from concentrate, which would allow the detection of fraudulently added mandarin juice. The possible use of the dual-probe system for quantitative measurements was also tested on fruit juice mixtures. qPCR data obtained from samples containing equal amounts of mandarin and orange juice revealed that the mandarin target copy number was approximately 2.6-fold higher than in orange juice. The use of a matrix-adapted control as calibrator to compensate the resulting C(T) bias allowed accurate quantitative measurements to be obtained. PMID:24128588

  18. Direct Detection of Cylindrocarpon destructans, Root Rot Pathogen of Ginseng by Nested PCR from Soil Samples.

    PubMed

    Jang, Chang Soon; Lim, Jin Ha; Seo, Mun Won; Song, Jeong Young; Kim, Hong Gi

    2010-03-01

    We have successfully applied the nested PCR to detect Cylindrocarpon destructans, a major pathogen causing root rot disease from ginseng seedlings in our former study. The PCR assay, in this study, was used to detect the pathogen from soils. The nested PCR using internal transcribed spacer (ITS) 1, 4 primer set and Dest 1, 4 primer set maintained the specificity in soils containing various microorganisms. For a soil DNA extraction method targeting chlamydospores, when several cell wall disrupting methods were tested, the combination of lyophilization and grinding with glass beads, which broke almost all the chlamydospores, was the strongest. The DNA extraction method which was completed based on the above was simple and time-saving because of exclusion of unnecessary stages, and efficient to apply in soils. As three ginseng fields whose histories were known were analyzed, the PCR assay resulted as our expectation derived from the field information. The direct PCR method will be utilized as a reliable and rapid tool for detecting and monitoring C. destructans in ginseng fields. PMID:23956622

  19. Innovative qPCR using interfacial effects to enable low threshold cycle detection and inhibition relief

    PubMed Central

    Harshman, Dustin K.; Rao, Brianna M.; McLain, Jean E.; Watts, George S.; Yoon, Jeong-Yeol

    2015-01-01

    Molecular diagnostics offers quick access to information but fails to operate at a speed required for clinical decision-making. Our novel methodology, droplet-on-thermocouple silhouette real-time polymerase chain reaction (DOTS qPCR), uses interfacial effects for droplet actuation, inhibition relief, and amplification sensing. DOTS qPCR has sample-to-answer times as short as 3 min 30 s. In infective endocarditis diagnosis, DOTS qPCR demonstrates reproducibility, differentiation of antibiotic susceptibility, subpicogram limit of detection, and thermocycling speeds of up to 28 s/cycle in the presence of tissue contaminants. Langmuir and Gibbs adsorption isotherms are used to describe the decreasing interfacial tension upon amplification. Moreover, a log-linear relationship with low threshold cycles is presented for real-time quantification by imaging the droplet-on-thermocouple silhouette with a smartphone. DOTS qPCR resolves several limitations of commercially available real-time PCR systems, which rely on fluorescence detection, have substantially higher threshold cycles, and require expensive optical components and extensive sample preparation. Due to the advantages of low threshold cycle detection, we anticipate extending this technology to biological research applications such as single cell, single nucleus, and single DNA molecule analyses. Our work is the first demonstrated use of interfacial effects for sensing reaction progress, and it will enable point-of-care molecular diagnosis of infections. PMID:26601245

  20. Rapid and sensitive detection of major uropathogens in a single-pot multiplex PCR assay.

    PubMed

    Padmavathy, B; Vinoth Kumar, R; Patel, Amee; Deepika Swarnam, S; Vaidehi, T; Jaffar Ali, B M

    2012-07-01

    Urinary tract infection (UTI) is among the most common bacterial infections and poses a significant healthcare burden. Escherichia coli is the most common cause of UTI accounting for up to 70 % and a variable contribution from Proteus mirabilis, Pseudomonas aeruginosa and Klebsiella pneumoniae. To establish a complete diagnostic system, we have developed a single-tube multiplex PCR assay (mPCR) for the detection of the above-mentioned four major uropathogens. The sensitivity of the assay was found to be as low as 10(2) cfu/ml of cells. The mPCR evaluated on 280 clinical isolates detected 100 % of E. coli, P. aeruginosa, P. mirabilis and 95 % of K. pneumonia. The assay was performed on 50 urine samples and found to be specific and sensitive for clinical diagnosis. In addition, the mPCR was also validated on spiked urine samples using 40 clinical isolates to demonstrate its application under different strain used in this assay. In total, mPCR reported here is a rapid and simple screening tool that can compete with conventional biochemical-based screening assays that may require 2-3 days for detection. PMID:22526571

  1. Innovative qPCR using interfacial effects to enable low threshold cycle detection and inhibition relief.

    PubMed

    Harshman, Dustin K; Rao, Brianna M; McLain, Jean E; Watts, George S; Yoon, Jeong-Yeol

    2015-09-01

    Molecular diagnostics offers quick access to information but fails to operate at a speed required for clinical decision-making. Our novel methodology, droplet-on-thermocouple silhouette real-time polymerase chain reaction (DOTS qPCR), uses interfacial effects for droplet actuation, inhibition relief, and amplification sensing. DOTS qPCR has sample-to-answer times as short as 3 min 30 s. In infective endocarditis diagnosis, DOTS qPCR demonstrates reproducibility, differentiation of antibiotic susceptibility, subpicogram limit of detection, and thermocycling speeds of up to 28 s/cycle in the presence of tissue contaminants. Langmuir and Gibbs adsorption isotherms are used to describe the decreasing interfacial tension upon amplification. Moreover, a log-linear relationship with low threshold cycles is presented for real-time quantification by imaging the droplet-on-thermocouple silhouette with a smartphone. DOTS qPCR resolves several limitations of commercially available real-time PCR systems, which rely on fluorescence detection, have substantially higher threshold cycles, and require expensive optical components and extensive sample preparation. Due to the advantages of low threshold cycle detection, we anticipate extending this technology to biological research applications such as single cell, single nucleus, and single DNA molecule analyses. Our work is the first demonstrated use of interfacial effects for sensing reaction progress, and it will enable point-of-care molecular diagnosis of infections. PMID:26601245

  2. Development and Application of Real-Time PCR for Detection of Subgroup J Avian Leukosis Virus

    PubMed Central

    Qin, Liting; Gao, Yulong; Ni, Wei; Sun, Meiyu; Wang, Yongqiang; Yin, Chunhong; Qi, Xiaole; Gao, Honglei

    2013-01-01

    Subgroup J avian leukosis virus (ALV-J) is an avian retrovirus that causes severe economic losses in the poultry industry. The early identification and removal of virus-shedding birds are important to reduce the spread of congenital and contact infections. In this study, a TaqMan-based real-time PCR method for the rapid detection and quantification of ALV-J with proviral DNA was developed. This method exhibited a high specificity for ALV-J. Moreover, the detection limit was as low as 10 viral DNA copies. The coefficients of variation (CVs) of both interassay and intra-assay reproducibility were less than 1%. The growth curves of ALV-J in DF-1 cells were measured by real-time PCR, yielding a trend line similar to those determined by 50% tissue culture infective dose (TCID50) and p27 antigen detection. Tissue samples suspected of ALV infection were evaluated using real-time PCR, virus isolation, and routine PCR, and the positivity rates were 60.1%, 41.6% and 44.5%, respectively. Our data indicated that the real-time PCR method provides a sensitive, specific, and reproducible diagnostic tool for the identification and quantification of ALV-J for clinical diagnosis and in laboratory research. PMID:23100340

  3. Development and evaluation of a new PCR assay for detection of Pseudomonas aeruginosa D genotype.

    PubMed

    Lødeng, A G G; Ahlén, C; Lysvand, H; Mandal, L H; Iversen, O J

    2006-08-01

    This report describes a new PCR-based assay for the detection of Pseudomonas aeruginosa genotype D in occupational saturation diving systems in the North Sea. This genotype has persisted in these systems for 11 years (1993-2003) and represents 18% of isolates from infections analysed during this period. The new PCR assay was based on sequences obtained after randomly amplified polymorphic DNA (RAPD)-PCR analysis of a group of isolates related to diving that had been identified previously by pulsed-field gel electrophoresis (PFGE). The primer set for the D genotype targets a gene that codes for a hypothetical class 4 protein in the P. aeruginosa PAO1 genome. A primer set able to detect P. aeruginosa at the species level was also designed, based on the 23S-5S rDNA spacer region. The two assays produced 382-bp and 192-bp amplicons, respectively. The PCR assay was evaluated by analysing 100 P. aeruginosa isolates related to diving, representing 28 PFGE genotypes, and 38 clinical and community P. aeruginosa isolates and strains from other species. The assay identified all of the genotype D isolates tested. Two additional diving-relevant genotypes (TP2 and TP27) were also identified, as well as three isolates of non-diving origin. It was concluded that the new PCR assay is a useful tool for early detection and prevention of infections with the D genotype. PMID:16842571

  4. PCR detection of thermophilic spore-forming bacteria involved in canned food spoilage.

    PubMed

    Prevost, S; Andre, S; Remize, F

    2010-12-01

    Thermophilic bacteria that form highly heat-resistant spores constitute an important group of spoilage bacteria of low-acid canned food. A PCR assay was developed in order to rapidly trace these bacteria. Three PCR primer pairs were designed from rRNA gene sequences. These primers were evaluated for the specificity and the sensitivity of detection. Two primer pairs allowed detection at the species level of Geobacillus stearothermophilus and Moorella thermoacetica/thermoautrophica. The other pair allowed group-specific detection of anaerobic thermophilic bacteria of the genera Thermoanaerobacterium, Thermoanaerobacter, Caldanerobium and Caldanaerobacter. After a single enrichment step, these PCR assays allowed the detection of 28 thermophiles from 34 cans of spoiled low-acid food. In addition, 13 ingredients were screened for the presence of these bacteria. This PCR assay serves as a detection method for strains able to spoil low-acid canned food treated at 55°C. It will lead to better reactivity in the canning industry. Raw materials and ingredients might be qualified not only for quantitative spore contamination, but also for qualitative contamination by highly heat-resistant spores. PMID:20397018

  5. Detecting and Genotyping Escherichia coli O157:H7 using multiplexed PCR and nucleic acid microarrays

    SciTech Connect

    Call, Douglas R.; Brockman, Fred J. ); Chandler, Darrell P.

    2000-12-01

    Rapid detection and characterization of food borne pathogens such as Escherichia coli O157:H7 is crucial for epidemiological investigations and food safety surveillance. As an alternative to conventional technologies, we examined the sensitivity and specificity of nucleic acid microarrays for detecting and genotyping E. coli O157:H7. The array was composed of oligonucleotide probes (25-30 mer) complementary to four virulence loci (intimin, Shiga-like toxins I and II, and hemolysin A). Target DNA was amplified from whole cells or from purified DNA via single or multiplexed polymerase chain reaction (PCR), and PCR products were hybridized to the array without further modification or purification. The array was 32-fold more sensitive than gel electrophoresis and capable of detecting amplification products from < 1 cell equivalent of genomic DNA (1 fg). Immunomagnetic capture, PCR and a microarray were subsequently used to detect 55 CFU ml-1 (E. coli O157:H7) from chicken rinsate without the aid of pre-enrichment. Four isolates of E. coli O157:H7 and one isolate of O91:H2, for which genotypic data were available, were unambiguously genotyped with this array. Glass based microarrays are relatively simple to construct and provide a rapid and sensitive means to detect multiplexed PCR products and the system is amenable to automation.

  6. Electrochemical DNA sensor for anthrax toxin activator gene atxA-detection of PCR amplicons.

    PubMed

    Das, Ritu; Goel, Ajay K; Sharma, Mukesh K; Upadhyay, Sanjay

    2015-12-15

    We report the DNA probe functionalized electrochemical genosensor for the detection of Bacillus anthracis, specific towards the regulatory gene atxA. The DNA sensor is fabricated on electrochemically deposited gold nanoparticle on self assembled layer of (3-Mercaptopropyl) trimethoxysilane (MPTS) on GC electrode. DNA hybridization is monitored by differential pulse voltammogram (DPV). The modified GC electrode is characterized by atomic force microscopy (AFM), cyclic voltammetry (CV), and electrochemical impedance spectroscopy (EIS) method. We also quantified the DNA probe density on electrode surface by the chronocoulometric method. The detection is specific and selective for atxA gene by DNA probe on the electrode surface. No report is available for the detection of B. anthracis by using atxA an anthrax toxin activator gene. In the light of real and complex sample, we have studied the PCR amplicons of 303, 361 and 568 base pairs by using symmetric and asymmetric PCR approaches. The DNA probe of atxA gene efficiently hybridizes with different base pairs of PCR amplicons. The detection limit is found to be 1.0 pM (S/N ratio=3). The results indicate that the DNA sensor is able to detect synthetic target as well as PCR amplicons of different base pairs. PMID:26257186

  7. Development of a PCR test system for specific detection of Salmonella Paratyphi B in foods.

    PubMed

    Zhai, Ligong; Yu, Qian; Bie, Xiaomei; Lu, Zhaoxin; Lv, Fengxia; Zhang, Chong; Kong, Xiaohan; Zhao, Haizhen

    2014-06-01

    Salmonella enterica serotype Paratyphi B is a globally distributed human-specific pathogen causing paratyphoid fever. The aim of this study was to develop a rapid and reliable polymerase chain reaction (PCR) assay for its detection in food. The SPAB_01124 gene was found to be unique to S. Paratyphi B using comparative genomics. Primers for fragments of the SPAB_01124 gene and the Salmonella-specific invA gene were used in combination to establish a multiplex PCR assay that showed 100% specificity across 45 Salmonella strains (representing 34 serotypes) and 18 non-Salmonella strains. The detection limit was 2.2 CFU mL(-1) of S. Paratyphi B after 12-h enrichment in pure culture. It was shown that co-culture with S. Typhimurium or Escherichia coli up to concentrations of 3.6 × 10(5)  CFU and 3.3 × 10(4)  CFU, respectively, did not interfere with PCR detection of S. Paratyphi B. In artificially contaminated milk, the assay could detect as few as 62 CFU mL(-1) after 8 h of enrichment. In conclusion, comparative genomics was found to be an efficient approach to the mining of pathogen-specific target genes, and the PCR assay that was developed from this provided a rapid, specific, and sensitive method for detection of S. Paratyphi B. PMID:24725227

  8. Detecting and genotyping Escherichia coli O157:H7 using multiplexed PCR and nucleic acid microarrays

    SciTech Connect

    Call, Douglas R.; Brockman, Fred J.; Chandler, Darrell P.

    2001-07-05

    Rapid detection and characterization of food borne pathogens such as Escherichia coli O157:H7 is crucial for epidemiological investigations and food safety surveillance. As an alternative to conventional technologies, we examined the sensitivity and specificity of nucleic acid microarrays for detecting and genotyping E. coli O157:H7. The array was composed of oligonucleotide probes (25-30 mer) complementary to four virulence loci (intimin, Shiga-like toxins I and II, and hemolysin A). Target DNA was amplified from whole cells or from purified DNA via single or multiplexed polymerase chain reaction (PCR), and PCR products were hybridized to the array without further modification or purification. The array was 32-fold more sensitive than gel electrophoresis and capable of detecting amplification products from < 1 cell equivalent of genomic DNA (1 fg). Immunomagnetic capture, PCR and a microarray were subsequently used to detect 55 CFUs ml-1 (E. coli O157:H7) from chicken rinsate without the aid of pre-enrichment. Four isolates of E. coli O157:H7 and one isolate of O91:H2, for which genotypic data were available, were unambiguously genotyped with this array. Glass based microarrays are relatively simple to construct and provide a rapid and sensitive means to detect multiplexed PCR products and the system is amenable to automation.

  9. Leishmania species: Detection and identification by nested PCR assay from skin samples of rodent reservoirs

    PubMed Central

    Akhavan, Amir Ahmad; Mirhendi, Hossein; Khamesipour, Ali; Alimohammadian, Mohammad Hossein; Rassi, Yavar; Bates, Paul; Kamhawi, Shaden; Valenzuela, Jesus G.; Arandian, Mohammad Hossein; Abdoli, Hamid; Jalali-zand, Niloufar; Jafari, Reza; Shareghi, Niloufar; Ghanei, Maryam; Yaghoobi-Ershadi, Mohammad Reza

    2010-01-01

    Many rodent species act as reservoir hosts of zoonotic cutaneous leishmaniasis in endemic areas. In the present study a simple and reliable assay based on nested PCR was developed for the detection and identification of Leishmania parasites from rodent skin samples. We designed Leishmania-specific primers that successfully amplified ITS regions of Leishmania major, Leishmania gerbilli and Leishmania turanica using nested PCR. Out of 95 field collected Rhombomys opimus, 21 were positive by microscopic examination and 48 by nested PCR. The percentage of gerbils infected with L. major, L. gerbilli and L. turanica was 3.2%, 1.1% and 27.4%, respectively. In 15.8% of the rodents, we found mixed natural infections by L. major and L. turanica, 1.1% by L. major and L. gerbilli, and 2.1% by the three species. We concluded that this method is simple and reliable for detecting and identifying Leishmania species circulating in rodent populations. PMID:20566364

  10. Optimisation of one-tube PCR-ELISA to detect femtogram amounts of genomic DNA.

    PubMed

    Wilson, T; Carson, J; Bowman, J

    2002-10-01

    A simple, high-throughput, low-cost polymerase chain reaction-enzyme linked immunosorbent assay (PCR-ELISA) protocol that detects the presence of 4 fg of DNA from four bacterial fish pathogens Yersinia ruckeri, Tenacibaculum maritimum (formerly Flexibacter maritimus), Lactococcus garvieae and Aeromonas salmonicida was developed. DNA amplification was undertaken in a biphasic system with free and bound PCR that are achieved in the one NucleoLink tube. Solid-phase amplicons were detected using biotin labelled hybridization probes and visualised colourimetrically with streptavidin-alkaline phosphatase and p-nitrophenylphosphate as substrate. PCR and hybridization took less than 8 h to perform with maximum signal output for femtogram amounts of template DNA achieved within 24 h. Implementation and optimization of the protocol is discussed. PMID:12133608

  11. Multiplex PCR-based detection of Leptospira in environmental water samples obtained from a slum settlement.

    PubMed

    Vital-Brazil, Juliana Magalhães; Balassiano, Ilana Teruszkin; Oliveira, Fabiano Sutter de; Costa, Alberto Dias de Souza; Hillen, Leandro; Pereira, Martha Maria

    2010-05-01

    The aim of this study was to apply a molecular protocol to detect leptospiral DNA in environmental water samples. The study was carried out in a peri-urban settlement in Petrópolis, state of Rio de Janeiro. A multiplex PCR method employing the primers LipL32 and 16SrRNA was used. Three out of 100 analysed samples were positive in the multiplex PCR, two were considered to have saprophytic leptospires and one had pathogenic leptospires. The results obtained supported the idea that multiplex PCR can be used to detect Leptospira spp in water samples. This method was also able to differentiate between saprophytic and pathogenic leptospires and was able to do so much more easily than conventional methodologies. PMID:20512254

  12. Detecting Polychlorinated Biphenyls by Ah Receptor and Fluorescence Quantitative PCR with Exonuclease

    NASA Astrophysics Data System (ADS)

    Zhao, Xiaoxiang; Zhuang, Huisheng

    2010-11-01

    Tetrachlorobiphenyls as ligands were cultivated with goldfish, Ah receptors were extracted from the liver of goldfish and purified by hydroxyapatite. The complex of TCB ligands-receptors were analyzed by Surface Plasmon Resonance. DNA probes were amplified by PCR using Primers F1 and F2 with the DNA recognition site of responsive enhancer. DNA probes bound to the complex were not digested by exonuclease. The DNA that bound to the complex was quantified by real time PCR. A standard curve with TCB concentration to Ct values was obtained in the range of 10-12mol/L to 10-8 mol/L, according to TCB concentration in samples. The detection limit of the assay was below 10-12mol/L of TCB. Compared with HPLC, this assay is much more sensitive. These results suggest that fluorescence quantitative PCR with exonuclease by Ah receptors fits for detection of trace PCB.

  13. [RT-PCR detecting NUP98-HOX fusion gene in leukemia].

    PubMed

    Zhang, Yan; Li, Ling; Wen, Bing-Zhao; Lin, Ren-Yong; Cao, Xu; Wang, Ning; Ha Li Da, Ya Seng; Jiang, Ming; Wen, Hao; Lu, Xiao-Mei; Feng, Xiao-Hui; Wang, Xin

    2005-02-01

    To investigate whether there are NUP98-HOXA, NUP98-HOXB, NUP98-HOXC, NUP98-HOXD fusion genes in leukemia patients in Xinjiang, cellular total RNA was extracted from the bone marrow mononuclear cells, the formaldehyde-agarose gel electrophoresis was used to judge whether RNA was intact, the 17 RT-PCR primers were designed to amplify the predicted fusion junctions and 412 bp GAPDH was used as an internal control, NUP98-HOXA fusion genes were amplified by nested-PCR following reverse transcription. One-step PCR was performed to amplify the other predicted fusion genes. The results showed that RNA was proved to be intact and expression of GAPDH was found in every sample. However, no predicted fusion transcripts were detected in leukemia patients. In conclusion, no NUP98-HOX fusion genes were detected in the samples from Xinjiang. PMID:15748441

  14. A novel method for the diagnosis of drowning by detection of Aeromonas sobria with PCR method.

    PubMed

    Aoyagi, Miwako; Iwadate, Kimiharu; Fukui, Kenji; Abe, Shuntaro; Sakai, Kentaro; Maebashi, Kyoko; Ochiai, Eriko; Nakamura, Mihoko

    2009-11-01

    The acid digestion method has been widely used for the diagnosis of death by drowning, but it is not always sensitive. However, there has been no definitive method to replace acid digestion until now. We speculate that bacteria are more useful markers than plankton for the diagnosis of death by drowning. In this study, from the preserved blood samples of 32 freshwater drowning cases, specific DNA fragments of Aeromonas sobria, one of the most common aquatic bacteria, were examined using PCR. The DNA fragments of the bacterium were detected from 27 of 32 cases with first round PCR or nested-PCR. The remaining 5 cases in which bacterial DNA was not detected had longer storage periods for the blood samples and shorter time intervals from drowning to death. These results indicate that the present method can be applied to the diagnosis of death by drowning. PMID:19766051

  15. Comparison of a PNA clamp PCR and an ARMS/Scorpion PCR assay for the detection of K-ras mutations.

    PubMed

    Nordgård, Oddmund; Oltedal, Satu; Janssen, Emiel A M; Gilje, Bjørnar; Kørner, Hartwig; Tjensvoll, Kjersti; Smaaland, Rune

    2012-03-01

    Point mutations in the K-ras gene have been shown to confer resistance against epidermal growth factor receptor-directed therapy of metastatic colorectal cancer. Accordingly, K-ras mutation testing has become mandatory in hospitals offering such treatment. We compared the performance and reagent costs of 2 sensitive methods for detection of K-ras mutations: a peptide nucleic acid (PNA) clamp polymerase chain reaction (PCR) assay and a commercially available amplification refractory mutation system/Scorpion (ARMS/S) PCR assay. Both methods were applied in parallel to 101 formalin-fixed, paraffin-embedded tumor and metastasis samples from patients with colon cancer. The PNA clamp PCR assay detected K-ras mutations in 35% (35 of 101) of the samples, whereas the ARMS/S PCR assay detected mutations in 27% (27 of 101) of them. There was 92% (93 of 101) concordance between the 2 methods and the κ coefficient for the comparison was 0.82. The 8 discordant cases were exclusively positive by PNA clamp PCR. Finally, the reagent costs of the PNA clamp PCR assay were estimated to be at least 20 times lower than the ARMS/S assay. We concluded that the high performance and low costs associated with the PNA clamp PCR assay encourage its use in the administration of personalized epidermal growth factor receptor-directed therapy. PMID:22306670

  16. Simultaneous Detection of Marine Fish Pathogens by Using Multiplex PCR and a DNA Microarray

    PubMed Central

    González, Santiago F.; Krug, Melissa J.; Nielsen, Michael E.; Santos, Ysabel; Call, Douglas R.

    2004-01-01

    We coupled multiplex PCR and a DNA microarray to construct an assay suitable for the simultaneous detection of five important marine fish pathogens (Vibrio vulnificus, Listonella anguillarum, Photobacterium damselae subsp. damselae, Aeromonas salmonicida subsp. salmonicida, and Vibrio parahaemolyticus). The array was composed of nine short oligonucleotide probes (25-mer) complementary to seven chromosomal loci (cyt, rpoN, gyrB, toxR, ureC, dly, and vapA) and two plasmid-borne loci (fatA and A.sal). Nine primer sets were designed to amplify short fragments of these loci (100 to 177 bp) in a multiplex PCR. PCR products were subsequently labeled by nick translation and hybridized to the microarray. All strains of the five target species (n = 1 to 21) hybridized to at least one species-specific probe. Assay sensitivities ranged from 100% for seven probes to 83 and 67% for the two remaining probes. Multiplex PCR did not produce any nonspecific amplification products when tested against 23 related species of bacteria (n = 40 strains; 100% specificity). Using purified genomic DNA, we were able to detect PCR products with <20 fg of genomic DNA per reaction (equivalent to four or five cells), and the array was at least fourfold more sensitive than agarose gel electrophoresis for detecting PCR products. In addition, our method allowed the tentative identification of virulent strains of L. anguillarum serotype O1 based on the presence of the fatA gene (67% sensitivity and 100% specificity). This assay is a sensitive and specific tool for the simultaneous detection of multiple pathogenic bacteria that cause disease in fish and humans. PMID:15070982

  17. Simultaneous detection of marine fish pathogens by using multiplex PCR and a DNA microarray.

    PubMed

    González, Santiago F; Krug, Melissa J; Nielsen, Michael E; Santos, Ysabel; Call, Douglas R

    2004-04-01

    We coupled multiplex PCR and a DNA microarray to construct an assay suitable for the simultaneous detection of five important marine fish pathogens (Vibrio vulnificus, Listonella anguillarum, Photobacterium damselae subsp. damselae, Aeromonas salmonicida subsp. salmonicida, and Vibrio parahaemolyticus). The array was composed of nine short oligonucleotide probes (25-mer) complementary to seven chromosomal loci (cyt, rpoN, gyrB, toxR, ureC, dly, and vapA) and two plasmid-borne loci (fatA and A.sal). Nine primer sets were designed to amplify short fragments of these loci (100 to 177 bp) in a multiplex PCR. PCR products were subsequently labeled by nick translation and hybridized to the microarray. All strains of the five target species (n = 1 to 21) hybridized to at least one species-specific probe. Assay sensitivities ranged from 100% for seven probes to 83 and 67% for the two remaining probes. Multiplex PCR did not produce any nonspecific amplification products when tested against 23 related species of bacteria (n = 40 strains; 100% specificity). Using purified genomic DNA, we were able to detect PCR products with < 20 fg of genomic DNA per reaction (equivalent to four or five cells), and the array was at least fourfold more sensitive than agarose gel electrophoresis for detecting PCR products. In addition, our method allowed the tentative identification of virulent strains of L. anguillarum serotype O1 based on the presence of the fatA gene (67% sensitivity and 100% specificity). This assay is a sensitive and specific tool for the simultaneous detection of multiple pathogenic bacteria that cause disease in fish and humans. PMID:15070982

  18. Evaluation and Field Validation of PCR Tests for Detection of Actinobacillus pleuropneumoniae in Subclinically Infected Pigs

    PubMed Central

    Fittipaldi, Nahuel; Broes, André; Harel, Josée; Kobisch, Marylène; Gottschalk, Marcelo

    2003-01-01

    Eight PCR tests were evaluated for their abilities to detect Actinobacillus pleuropneumoniae in swine tonsils. At first they were compared regarding their specificities by using A. pleuropneumoniae and related bacterial species and their analytical sensitivities by using tonsils experimentally infected in vitro. PCRs were carried out both directly with tonsil homogenates (direct PCR) and after culture of the sample (after-culture PCR). Most tests demonstrated good specificities; however, some tests gave false-positive results with some non-A. pleuropneumoniae species. High degrees of variation in the analytical sensitivities among the tests were observed for the direct PCRs (109 to 102 CFU/g of tonsil), whereas those of most of the after-culture PCRs were similar (102 CFU/g of tonsil). In a second phase, the effects of sample storage time and storage conditions were evaluated by using tonsils from experimentally infected animals. Storage at −20°C allowed the detection of the organism for at least 4 months. Finally, the omlA PCR test described by Savoye et al. (C. Savoye et al., Vet. Microbiol. 73:337-347, 2000) and the commercially available Adiavet App PCR test were further validated with field samples. Their effectiveness was compared to those of standard and immunomagnetic separation-based methods of bacterial isolation. In addition, a comparison of tonsil biopsy specimens (from living animals) and whole tonsils (collected at the slaughterhouse) was also conducted. A. pleuropneumoniae was neither isolated nor detected by PCR from a herd serologically negative for A. pleuropneumoniae. PCR was more sensitive than the standard isolation method with whole tonsils from three infected herds. After-culture PCR offered the highest degree of sensitivity (93 and 83% for the omlA and Adiavet App PCRs, respectively). The PCR detection rate was higher with whole tonsils than with tonsil biopsy specimens. Good agreement (κ = 0.65) was found between the presence of A

  19. Performance of PCR-based and Bioluminescent assays for mycoplasma detection.

    PubMed

    Falagan-Lotsch, Priscila; Lopes, Talíria Silva; Ferreira, Nívea; Balthazar, Nathália; Monteiro, Antônio M; Borojevic, Radovan; Granjeiro, José Mauro

    2015-11-01

    Contaminated eukaryotic cell cultures are frequently responsible for unreliable results. Regulatory entities request that cell cultures must be mycoplasma-free. Mycoplasma contamination remains a significant problem for cell cultures and may have an impact on biological analysis since they affect many cell parameters. The gold standard microbiological assay for mycoplasma detection involves laborious and time-consuming protocols. PCR-based and Bioluminescent assays have been considered for routine cell culture screening in research laboratories since they are fast, easy and sensitive. Thus, the aim of this work is to compare the performance of two popular commercial assays, PCR-based and Bioluminescent assays, by assessing the level of mycoplasma contamination in cell cultures from Rio de Janeiro Cell Bank (RJCB) and also from customers' laboratories. The results obtained by both performed assays were confirmed by scanning electron microscopy. In addition, we evaluated the limit of detection of the PCR kit under our laboratory conditions and the storage effects on mycoplasma detection in frozen cell culture supernatants. The performance of both assays for mycoplasma detection was not significantly different and they showed very good agreement. The Bioluminescent assay for mycoplasma detection was slightly more dependable than PCR-based due to the lack of inconclusive results produced by the first technique, especially considering the ability to detect mycoplasma contamination in frozen cell culture supernatants. However, cell lines should be precultured for four days or more without antibiotics to obtain safe results. On the other hand, a false negative result was obtained by using this biochemical approach. The implementation of fast and reliable mycoplasma testing methods is an important technical and regulatory issue and PCR-based and Bioluminescent assays may be good candidates. However, validation studies are needed. PMID:26296900

  20. From synthetic DNA to PCR product: detection of fungal infections using SERS.

    PubMed

    Mabbott, Samuel; Thompson, David; Sirimuthu, Narayana; McNay, Graeme; Faulds, Karen; Graham, Duncan

    2016-06-23

    We report the use of silver hydroxylamine nanoparticles functionalised with single stranded monothiolated DNA for the detection of fungal infections. The four different species of fungi that were targeted were Candida albicans, Candida glabrata, Candida krusei and Aspergillus fumigatus. Rational design of synthetic targets and probes was carried out by carefully analysing the 2-D folding of the DNA and then by global alignment of the sequences to ensure specificity. The effects of varying the concentrations of the DNA and dye surrounding the nanoparticles on the resultant surface enhanced Raman scattering (SERS) signal were also investigated to ensure compatibility of the probes in a multiplexed environment. Using principal components analysis (PCA) it was possible to detect the individual presence of each target and group them accordingly. The move to detect the C. krusei single stranded PCR product (ssPCR) was significant to demonstrate that the methodology could be employed for the detection and diagnosis of invasive fungal infections (IFDs) within a clinical setting. Initially the PCR product was subjected to an alkali shock method in order to separate the strands ready for detection using the nanoparticle probes system. This time 18 base probes were employed to enhance hybridisation efficiency and dextran sulfate was found to have a vital role in ensuring that detection of the C. krusei target was achieved. This demonstrated the use of DNA functionalised silver nanoparticle for the detection of clinically relevant DNA relating to a specific fungal infection and offers significant promise for future diagnostic applications. PMID:27034997

  1. Detection of bovine group a rotavirus using rapid antigen detection kits, rt-PCR and next-generation DNA sequencing.

    PubMed

    Minami-Fukuda, Fujiko; Nagai, Makoto; Takai, Hikaru; Murakami, Toshiaki; Ozawa, Tadashi; Tsuchiaka, Shinobu; Okazaki, Sachiko; Katayama, Yukie; Oba, Mami; Nishiura, Naomi; Sassa, Yukiko; Omatsu, Tsutomu; Furuya, Tetsuya; Koyama, Satoshi; Shirai, Junsuke; Tsunemitsu, Hiroshi; Fujii, Yoshiki; Katayama, Kazuhiko; Mizutani, Tetsuya

    2013-12-30

    We investigated the sensitivity of human rotavirus rapid antigen detection (RAD) kits, RT-PCR and next-generation DNA sequencing (NGS) for detection of bovine group A rotavirus (RVA). The Dipstick 'Eiken' Rota (Dipstick) showed the highest sensitivity out of the seven RAD kits against all selected strains in limited dilution analyses, which was consistent with the results for equine rotavirus previously reported. RT-PCR had 10⁰-10³-fold higher sensitivity than the Dipstick. NGS using thirteen RT-PCR-negative fecal samples revealed that all samples yielded RVA reads and especially that two of them covered all 11 genome segments. Moreover, mapping reads to reference sequences allowed genotyping. The NGS would be sensitive and useful for analysis of less dependent on specific primers and screening of genotypes. PMID:23912876

  2. Minimally Invasive Detection of Piscirickettsia salmonis in Cultivated Salmonids via the PCR

    PubMed Central

    Marshall, Sergio; Heath, Sekou; Henríquez, Vitalia; Orrego, Cristián

    1998-01-01

    The attributes of the PCR allowed implementation of an assay for specific detection of Piscirickettsia salmonis from a few microliters of fish serum. This opens the way to less invasive modes of sampling for this microbial pathogen in salmonids. PMID:9687475

  3. Single-tube, noninterrupted reverse transcription-PCR for detection of infectious bursal disease virus.

    PubMed Central

    Lee, L H; Ting, L J; Shien, J H; Shieh, H K

    1994-01-01

    An assay protocol based on single-tube, noninterrupted reverse transcription-PCR (RT-PCR) for the detection of infectious bursal disease virus (IBDV) is described. After the conditions for RT-PCR had been optimized, a primer set framing a region within the gene coding for IBDV VP2 protein was used to amplify a 318-bp fragment of the IBDV genome. Amplified product was detected with three strains of IBDV, whereas none was obtained from uninfected bursal tissue or seven unrelated avian viruses. The sensitivity of this RT-PCR was tested with purified viral RNA from three strains of IBDV. The detection limit was 10 fg in an ethidium bromide-stained gel. In addition, this assay system was used to detect IBDV in bursal-tissue specimens from commercially reared chickens. The identity of the amplified products from the tissue specimen preparation was determined by using a rapid, simple procedure in which internally nested, end-labeled probes were used. Images PMID:8051255

  4. International collaborative study to compare reverse transcriptase PCR assays for detection and genotyping of noroviruses.

    PubMed

    Vinjé, Jan; Vennema, Harry; Maunula, Leena; von Bonsdorff, Carl-Henrik; Hoehne, Marina; Schreier, Eckart; Richards, Alison; Green, Jon; Brown, David; Beard, Suzanne S; Monroe, Stephan S; de Bruin, Erwin; Svensson, Lennart; Koopmans, Marion P G

    2003-04-01

    To allow more rapid and internationally standardized assessment of the spread of noroviruses (previously called Norwalk-like viruses [NLVs]) as important food-borne pathogens, harmonization of methods for their detection is needed. Diagnosis of NLVs in clinical diagnostic laboratories is usually performed by reverse transciptase PCR (RT-PCR) assays. In the present study, the performance of five different RT-PCR assays for the detection of NLVs was evaluated in an international collaborative study by five laboratories in five countries with a coded panel of 91 fecal specimens. The assays were tested for their sensitivity, detection limit, and ease of standardization. In total, NLVs could be detected by at least one RT-PCR assay in 69 (84%) of the samples that originally tested positive. Sensitivity ranged from 52 to 73% overall and from 54 to 100% and 58 to 85% for genogroup I and II viruses, respectively. In all, 64% of the false-negative results were obtained with a set of diluted stools (n = 20) that may have lost quality upon storage. Sensitivity was improved when these samples were excluded from analysis. No one single assay stood out as the best, although the p1 assay demonstrated the most satisfactory overall performance. To promote comparability of data, this assay will be recommended for newly starting groups in future collaborative studies. PMID:12682125

  5. DETECTION OF RENIBACTERIUM SALMONINARUM IN CHINOOK SALMON ONCORHYNCHUS TSHAWYTSCHA USING QUANTITATIVE PCR.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We have developed a quantitative PCR assay to detect varying levels of Renibacterium salmoninarum, the causative agent of bacterial kidney disease (BKD). This assay allows for the direct enumeration of bacterial DNA or RNA copy number within tissues and body fluids. The assay can be applied nonletha...

  6. DETECTION AND QUANTITATION OF SOLENOPSIS INVICTA VIRUS IN FIRE ANTS BY REAL-TIME PCR

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A quantitative real-time PCR (QPCR) method was developed to detect and quantify the amount of Solenopsis invicta virus (SINV) infecting individual ants of Solenopsis invicta. The two-step method utilized a gene-specific oligonucleotide primer targeting the SINV RNA-dependent RNA polymerase (RdRp) f...

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

    PubMed

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

    2016-07-01

    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

  8. Generic RT-PCR tests for detection and identification of tospoviruses.

    PubMed

    Hassani-Mehraban, A; Westenberg, M; Verhoeven, J T J; van de Vossenberg, B T L H; Kormelink, R; Roenhorst, J W

    2016-07-01

    A set of tests for generic detection and identification of tospoviruses has been developed. Based on a multiple sequence alignment of the nucleocapsid gene and its 5' upstream untranslated region sequence from 28 different species, primers were designed for RT-PCR detection of tospoviruses from all recognized clades, i.e. the American, Asian and Eurasian clades, and from the small group of distinct and floating species. Pilot experiments on isolates from twenty different species showed that the designed primer sets successfully detected all species by RT-PCR, as confirmed by nucleotide sequence analysis of the amplicons. In a final optimized design, the primers were applied in a setting of five RT-PCR tests. Seven different tospoviruses were successfully identified from diagnostic samples and in addition a non-described tospovirus species from alstroemeria plants. The results demonstrate that the newly developed generic RT-PCR tests provide a relevant tool for broad detection and identification of tospoviruses in plant quarantine and diagnostic laboratories. PMID:27036502

  9. 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

  10. Preclinical Assessment of a Fully Automated Multiplex PCR Panel for Detection of Central Nervous System Pathogens.

    PubMed

    Hanson, K E; Slechta, E S; Killpack, J A; Heyrend, C; Lunt, T; Daly, J A; Hemmert, A C; Blaschke, A J

    2016-03-01

    We evaluated a multiplexed PCR panel for the detection of 16 bacterial, viral, and fungal pathogens in cerebrospinal fluid. Panel results were compared to routine testing, and discrepancies were resolved by additional nucleic acid amplification tests or sequencing. Overall, the positive and negative agreements across methods were 92.9% and 91.9%, respectively. PMID:26719436

  11. DETECTION OF PHAKOPSORA PACHYRHIZI SPORES IN RAIN USING REAL-TIME PCR ASSAY

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In 2005, rain samples were collected weekly at selected National Atmospheric Deposition Program (NADP) sites in the eastern and central US and screened for Phakopsora pachyrhizi (Asian soybean rust) spores. A nested real-time PCR assay was used to detect P. pachyrhizi DNA in the filter residue. A su...

  12. Single Laboratory Comparison of Host-Specific PCR Assays for the Detection of Bovine Fecal Pollution

    EPA Science Inventory

    There are numerous PCR-based methods available to detect bovine fecal pollution in ambient waters. Each method targets a different gene and microorganism leading to differences in method performance, making it difficult to determine which approach is most suitable for field appl...

  13. Towards Q-PCR of pathogenic bacteria with improved electrochemical double-tagged genosensing detection

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A very sensitive assay for the rapid detection of pathogenic bacteria based on electrochemical genosensing has been designed. The assay was performed by the PCR specific amplification of the eaeA gene, related with the pathogenic activity of Escherichia coli O157:H7. The efficiency and selectivity o...

  14. DEVELOPMENT OF A REAL-TIME FLUORESCENCE RESONANCE ENERGY TRANSFER (FRET) PCR TO DETECT ARCOBACTER SPECIES

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A real-time PCR targeting the gyrase A subunit gene outside the quinolone resistance-determining region has been developed to detect Arcobacter species. The species identification was made by probe hybridization and melting curve analysis, using the Fluorescence Resonance Energy Transfer technology...

  15. DEVELOPMENT OF A REAL-TIME FLUORESCENCE RESONANCE ENERGY TRANSFER PCR TO DETECT ARCOBACTER SPECIES

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A real-time PCR targeting the gyrase A subunit gene outside the quinolone resistance-determining region has been developed to detect Arcobacter species. The species identification was made by probe hybridization and melting curve analysis, using Fluorescence Resonance Energy Transfer technology. D...

  16. 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…

  17. Detection of porcine reproductive and respiratory syndrome virus in boar semen by PCR.

    PubMed

    Christopher-Hennings, J; Nelson, E A; Nelson, J K; Hines, R J; Swenson, S L; Hill, H T; Zimmerman, J J; Katz, J B; Yaeger, M J; Chase, C C

    1995-07-01

    Porcine reproductive and respiratory syndrome virus (PRRSV) causes a devastating disease in swine. The presence and transmission of PRRSV by boar semen has been demonstrated by using a swine bioassay. In this assay, 4- to 8-week-old pigs were inoculated intraperitoneally with semen from PRRSV-infected boars. Seroconversion of these piglets indicated the presence of PRRSV in semen. Seroconversion in gilts has also been demonstrated following artificial insemination with semen from PRRSV-infected boars. These methods of detecting PRRSV in boar semen are time-consuming, laborious, and expensive. The objective of this study was to develop a reliable and sensitive PCR assay to directly detect PRRSV in boar semen. Primers from open reading frames 1b and 7 of the PRRSV genome were used in nested PCRs. Virus was detected at concentrations as low as 10 infectious virions per ml in PRRSV-spiked semen. Specificity was confirmed by using a nested PCR and a 32P-labeled oligonucleotide probe. The primers did not react with related arteriviruses or other swine viruses. The PCR assay showed good correlation with the swine bioassay, and both methods were superior to virus isolation. To consistently identify PRRSV in boar semen, the cell fraction was separated by centrifugation at 600 x g for 20 min, a lysis buffer without a reducing agent (2-mercaptoethanol) was used, and nondiluted and 1:20-diluted cell fractions were evaluated by PCR. PRRSV was not reliably detected in the seminal plasma fraction of boar semen. PMID:7665637

  18. Effective detection of human adenovirus in hawaiian waters using enhanced pcr methods

    PubMed Central

    2011-01-01

    Background The current criteria for recreational water quality evaluation are primarily based on measurements of fecal indicator bacteria growth. However, these criteria often fail to predict the presence of waterborne human pathogenic viruses. To explore the possibility of direct use of human enteric viruses as improved human fecal contamination indicators, human adenovirus (HAdV) was tested as a model in this study. Findings In order to establish a highly sensitive protocol for effective detection of HAdV in aquatic environments, sixteen published PCR primer sets were re-optimized and comparatively evaluated. Primer sets nehex3deg/nehex4deg, ADV-F/ADV-R, and nested PCR primer sets hex1deg/hex2deg and nehex3deg/nehex4deg were identified to be the most sensitive ones, with up to 1,000 fold higher detection sensitivity compared to other published assays. These three PCR protocols were successfully employed to detect HAdV in both treated and untreated urban wastewaters, and also in 6 of 16 recreational water samples collected around the island of Oahu, Hawaii. Conclusions Findings from this study support the possible use of enteric viruses for aquatic environmental monitoring, specifically for the essential routine monitoring of Hawaiian beach waters using the optimized PCR protocol to detect HAdV at certain water sites to ensure a safe use of recreational waters. PMID:21303549

  19. Specific Detection of Fusarium Species in Blood and Tissues by a PCR Technique

    PubMed Central

    Hue, Francois-Xavier; Huerre, Michel; Rouffault, Marie Ange; de Bievre, Claude

    1999-01-01

    Fusarium species are opportunistic nosocomial pathogens that often cause fatal invasive mycoses. We designed a primer pair that amplifies by PCR a fragment of a gene coding for the rRNA of Fusarium species. The DNAs of the main Fusarium species and Neocosmospora vasinfecta but not the DNAs from 11 medically important fungi were amplified by these primers. The lower limit of detection of the PCR system was 10 fg of Fusarium solani DNA by ethidium bromide staining. To test the ability of this PCR system to detect Fusarium DNA in tissues, we developed a mouse model of disseminated fusariosis. Using the PCR, we detected Fusarium DNA in mouse tissues and in spiked human blood. Furthermore, F. solani, Fusarium moniliforme, and Fusarium oxysporum were testing by random amplified polymorphic DNA (RAPD) analysis. The bands produced by RAPD analysis were purified, cloned, and sequenced. The information was used to design primer pairs that selectively amplified one or several Fusarium species. The method developed may be useful for the rapid detection and identification of Fusarium species both from culture and from clinical samples. PMID:10405380

  20. Quantitative PCR for Detection and Enumeration of Genetic Markers of Bovine Fecal Pollution

    EPA Science Inventory

    Accurate assessment of health risks associated with bovine (cattle) fecal pollution requires a reliable host-specific genetic marker and a rapid quantification method. We report the development of quantitative PCR assays for the detection of two recently described cow feces-spec...

  1. Detection of shrimp-derived components in food by real-time fluorescent PCR.

    PubMed

    Cao, Jijuan; Yu, Bing; Ma, Lidan; Zheng, Qiuyue; Zhao, Xin; Xu, Junyi

    2011-10-01

    Crustaceans such as shrimp and crabs and their products are important allergens in food, and allergic reactions due to the consumption of shrimp and crabs are frequently reported. However, the chemical properties of shrimp-derived allergens, except for Pen a I, are still unclear. Therefore, it is important to establish a more sensitive and specific method for detecting the composition of foods containing shrimp. In the present study, we developed a real-time fluorescent PCR to identify the specific shrimp-derived components in food. The primers and TaqMan probes for real-time fluorescent PCR were designed based on 16S rRNA genes through comparing a large number of nucleic acid sequences from different species of shrimp that have been published by the National Center for Biotechnology Information. In total, 56 kinds of samples, including different kinds of shrimp, crab, fish, shellfish, and octopus, were subjected to detection by real-time PCR. The results indicated that real-time fluorescent PCR could successfully identify the shrimp-derived components. In order to explore the effect of food processing on detection sensitivity, fish powder containing shrimp powder was treated by heating at 133°C for 30 min. The limit of detection of shrimp-derived components in fish powder was 0.05% (wt/wt). PMID:22004830

  2. Single laboratory comparison of host-specific PCR assays for the detection of bovine fecal pollution

    Technology Transfer Automated Retrieval System (TEKTRAN)

    There are numerous PCR-based methods available to detect bovine fecal pollution in ambient waters. Each method targets a different gene and microorganism leading to differences in method performance, making it difficult to determine which approach is most suitable for field applications. We descri...

  3. Detection of Cryptosporidium parvum in raw milk by PCR and oligonucleotide probe hybridization.

    PubMed Central

    Laberge, I; Ibrahim, A; Barta, J R; Griffiths, M W

    1996-01-01

    Cryptosporidium spp. are potential contaminants of food. Suspected cases of food-borne cryptosporidiosis are rarely confirmed because of the limited numbers of oocysts in the samples and the lack of sensitive detection methods adaptable to food. PCR was investigated as a means of overcoming this problem. A PCR assay was designed for the specific amplification of a previously sequenced portion of an oocyst protein gene fragment of Cryptosporidium parvum (N. C. Lally, G. D. Baird, S. J. McQuay, F. Wright, and J. J. Oliver, Mol. Biochem. Parasitol. 56:69-78, 1992) and compared with the primer set of Laxer et al. (M. A. Laxer, B. K. Timblin, and R. J. Patel, Am. J. Trop. Med. Hyg. 45:688-694, 1991). The PCR products were hybridized with digoxigenin-labeled internal probes and detected by chemiluminescence to enhance sensitivity. The two sets of primers were compared with regard to their sensitivity and specificity by using a variety of human and animal isolates of C. parvum and related parasites. Both assays enabled the detection of 1 to 10 oocysts in 20 ml of artificially contaminated raw milk. The assay based on the PCR set and probe of Laxer et al. detected DNAs from Eimeria acervulina and Giardia intestinalis. The new assay has good specificity for C. parvum bovine isolates and hence has a better potential for monitoring the prevalence of C. parvum in raw milk and other environmental samples. PMID:8795214

  4. Rapid Detection of Enterohemorrhagic Escherichia coli by Real-Time PCR with Fluorescent Hybridization Probes

    PubMed Central

    Bellin, Tobias; Pulz, Matthias; Matussek, Andreas; Hempen, Hans-Günther; Gunzer, Florian

    2001-01-01

    In this report, we present a PCR protocol for rapid identification of enterohemorrhagic Escherichia coli on a LightCycler instrument. In a multiplex assay, the genes encoding Shiga toxin 1 and Shiga toxin 2 are detected in a single reaction capillary. A complete analysis of up to 32 samples takes about 45 min. PMID:11136804

  5. Rapid detection method for fusaric acid-producing species of Fusarium by PCR

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Fusaric acid is a mycotoxin produced by species of the fungus Fusarium and can act synergistically with other Fusarium toxins. In order to develop a specific detection method for fusaric acid-producing fungus, PCR prim¬ers were designed to amplify FUB10, a transcription factor gene in fusaric acid ...

  6. An in-house multiplex pcr method to detect of putative virulence factors in aeromonas species

    PubMed Central

    Aguilera-Arreola, Ma. Guadalupe; Martínez, Alma Aidee Carmona; Castro-Escarpulli, Graciela

    2011-01-01

    A pentaplex PCR was developed and optimised to detect the genes that encode the five most important putative virulence factors in Aeromonas isolates. It seems to be more efficient than previously reported techniques and promises to be a powerful tool for more accurate risk assessments and for monitoring pathogenic strains. PMID:24031758

  7. Application of quantitative PCR assays to detection of human Bacteroides species in the intestines of pigs

    Technology Transfer Automated Retrieval System (TEKTRAN)

    When evaluating the efficacy of probiotic bacteria, it is beneficial to know whether a fed bacterium reaches the appropriate location in the digestive tract. Use of quantitative PCR could detect specific bacteria in a sample with a complex microbial community. In order to determine whether three Bac...

  8. PCR-specific detection of recently described Lotmaria passim (Trypanosomatidae) in Chilean apiaries.

    PubMed

    Arismendi, Nolberto; Bruna, Alex; Zapata, Nelson; Vargas, Marisol

    2016-02-01

    The recently described trypanosome Lotmaria passim is currently considered the most predominant trypanosomatid in honey bees worldwide and could be a factor in honey bee declines. For a specific and quick detection of this pathogen, we developed primers based on the SSU rRNA and gGAPDH genes for the detection of L. passim in Chilean honey beehives. PCR products amplified and sequenced for these primers shared 99-100% identity with other sequences of L. passim. The designed primers were specific and we were able to detect a high prevalence (40-90%) of L. passim in bee hives distributed throughout Chile. Our described PCR-based method offers a feasible and specific detection of L. passim in any honey bee samples. PMID:26721451

  9. Detection of MET Gene Copy Number in Cancer Samples Using the Droplet Digital PCR Method

    PubMed Central

    Zhang, Yanni; Tang, En-Tzu; Du, Zhiqiang

    2016-01-01

    Purpose The analysis of MET gene copy number (CN) has been considered to be a potential biomarker to predict the response to MET-targeted therapies in various cancers. However, the current standard methods to determine MET CN are SNP 6.0 in the genomic DNA of cancer cell lines and fluorescence in situ hybridization (FISH) in tumor models, respectively, which are costly and require advanced technical skills and result in relatively subjective judgments. Therefore, we employed a novel method, droplet digital PCR (ddPCR), to determine the MET gene copy number with high accuracy and precision. Methods The genomic DNA of cancer cell lines or tumor models were tested and compared with the MET gene CN and MET/CEN-7 ratio determined by SNP 6.0 and FISH, respectively. Results In cell lines, the linear association of the MET CN detected by ddPCR and SNP 6.0 is strong (Pearson correlation = 0.867). In tumor models, the MET CN detected by ddPCR was significantly different between the MET gene amplification and non-amplification groups according to FISH (mean: 15.4 vs 2.1; P = 0.044). Given that MET gene amplification is defined as MET CN >5.5 by ddPCR, the concordance rate between ddPCR and FISH was 98.0%, and Cohen's kappa coefficient was 0.760 (95% CI, 0.498–1.000; P <0.001). Conclusions The results demonstrated that the ddPCR method has the potential to quantify the MET gene copy number with high precision and accuracy as compared with the results from SNP 6.0 and FISH in cancer cell lines and tumor samples, respectively. PMID:26765781

  10. Continuous flow real-time PCR device using multi-channel fluorescence excitation and detection.

    PubMed

    Hatch, Andrew C; Ray, Tathagata; Lintecum, Kelly; Youngbull, Cody

    2014-02-01

    High throughput automation is greatly enhanced using techniques that employ conveyor belt strategies with un-interrupted streams of flow. We have developed a 'conveyor belt' analog for high throughput real-time quantitative Polymerase Chain Reaction (qPCR) using droplet emulsion technology. We developed a low power, portable device that employs LED and fiber optic fluorescence excitation in conjunction with a continuous flow thermal cycler to achieve multi-channel fluorescence detection for real-time fluorescence measurements. Continuously streaming fluid plugs or droplets pass through tubing wrapped around a two-temperature zone thermal block with each wrap of tubing fluorescently coupled to a 64-channel multi-anode PMT. This work demonstrates real-time qPCR of 0.1-10 μL droplets or fluid plugs over a range of 7 orders of magnitude concentration from 1 × 10(1) to 1 × 10(7). The real-time qPCR analysis allows dynamic range quantification as high as 1 × 10(7) copies per 10 μL reaction, with PCR efficiencies within the range of 90-110% based on serial dilution assays and a limit of detection of 10 copies per rxn. The combined functionality of continuous flow, low power thermal cycling, high throughput sample processing, and real-time qPCR improves the rates at which biological or environmental samples can be continuously sampled and analyzed. PMID:24297040

  11. Detection of Salmonella in Blood by PCR using iroB gene

    PubMed Central

    Harish, Belgode Narasimha; Menezes, Godfred Antony; Parija, Subash Chandra

    2014-01-01

    Background:Salmonella, a genus of more than 2500 serological variants (serovars), includes many organisms that can cause human disease. Enteric fever remains an important public health problem in developing countries. Non typhoidal Salmonella generally produce a self limited gastroenteritis in healthy individuals whereas in extremes of age and immunocompromised cause severe fatal disease. The protean manifestations make this disease a true diagnostic challenge. Aim: The present study was carried out to optimize PCR for detecting the Salmonella genus using iroB gene and evaluate its use in the rapid diagnosis of typhoid and non typhoidal salmonellosis. Materials and Methods: The study was carried out between August 2009 and July 2011 on blood samples from patients attending JIPMER hospital, Pondicherry, India with clinical suspicion of enteric fever and salmonellosis. Whole blood was used DNA extraction and conventional PCR done with iroB and fliC primers. Blood culture and Widal test were performed for all the patients. Statistical Analysis: Performed using Fischer’s exact test with Graphpad Instat 3. Results: PCR results were compared with blood culture. Sensitivity and specificity of PCR with fliC gene are 95.6% and 93.3% respectively. Sensitivity and specificity of PCR with iroB gene are 96.6% and 93.3% respectively Conclusion: With iroB gene, additional cases of Salmonella Paratyphi A and non typhoidal Salmonella were detected when compared to fliC gene. PMID:25584214

  12. Detection and identification of Malassezia species in domestic animals and aquatic birds by PCR-RFLP

    PubMed Central

    Zia, M.; Mirhendi, H.; Toghyani, M.

    2015-01-01

    The present study aimed at detection and species-level identification of the Malassezia yeasts in domestic animals and aquatic birds by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Samples were collected using tape strips and swabs from 471 animals including 97 horses, 102 cattle, 105 sheep, 20 camels, 60 dogs, 30 cats, 1 hamster, 1 squirrel, 50 aquatic birds and 5 turkeys. Tape-strip samples were examined by direct microscopy. All samples were inoculated on modified Leeming and Notman agar medium. DNA extracted from the yeast colonies was amplified by PCR using primers specific for 26S rDNA. RFLP of the PCR products was performed using Hin6I enzyme, and PCR and RFLP products were visualized by agarose gel electrophoresis. Malassezia yeasts were detected at the following frequencies: 15.46% in horses, 12.74% in cattle, 12.38% in sheep, 28.33% in dogs, 26.66% in cats and 26% in aquatic birds. Eighty colonies of 6 species were isolated: Malassezia globosa 41.25%, Malassezia furfur 22.5%, Malassezia restricta 15%, Malassezia sympodialis 15%, Malassezia pachydermatis 5% and Malassezia slooffiae 1.25%. Therefore different lipophilic Malassezia species are found in a wide diversity of animals and aquatic birds. PCR-RFLP is a suitable technique for identification of different Malassezia species. PMID:27175148

  13. Real-Time RT-PCR for the Detection of Lyssavirus Species

    PubMed Central

    Deubelbeiss, A.; Zahno, M.-L.; Zanoni, M.; Bruegger, D.; Zanoni, R.

    2014-01-01

    The causative agents of rabies are single-stranded, negative-sense RNA viruses in the genus Lyssavirus of Rhabdoviridae, consisting of twelve classified and three as yet unclassified species including classical rabies virus (RABV). Highly neurotropic RABV causes rapidly progressive encephalomyelitis with nearly invariable fatal outcome. Rapid and reliable diagnosis of rabies is highly relevant for public and veterinary health. Due to growing variety of the genus Lyssavirus observed, the development of suitable molecular assays for diagnosis and differentiation is challenging. This work focused on the establishment of a suitable real-time RT-PCR technique for rabies diagnosis as a complement to fluorescent antibody test and rabies tissue culture infection test as gold standard for diagnosis and confirmation. The real-time RT-PCR was adapted with the goal to detect the whole spectrum of lyssavirus species, for nine of which synthesized DNA fragments were used. For the detection of species, seven probes were developed. Serial dilutions of the rabies virus strain CVS-11 showed a 100-fold higher sensitivity of real-time PCR compared to heminested RT-PCR. Using a panel of thirty-one lyssaviruses representing four species, the suitability of the protocol could be shown. Phylogenetic analysis of the sequences obtained by heminested PCR allowed correct classification of all viruses used. PMID:26464934

  14. Real-Time RT-PCR for the Detection of Lyssavirus Species.

    PubMed

    Deubelbeiss, A; Zahno, M-L; Zanoni, M; Bruegger, D; Zanoni, R

    2014-01-01

    The causative agents of rabies are single-stranded, negative-sense RNA viruses in the genus Lyssavirus of Rhabdoviridae, consisting of twelve classified and three as yet unclassified species including classical rabies virus (RABV). Highly neurotropic RABV causes rapidly progressive encephalomyelitis with nearly invariable fatal outcome. Rapid and reliable diagnosis of rabies is highly relevant for public and veterinary health. Due to growing variety of the genus Lyssavirus observed, the development of suitable molecular assays for diagnosis and differentiation is challenging. This work focused on the establishment of a suitable real-time RT-PCR technique for rabies diagnosis as a complement to fluorescent antibody test and rabies tissue culture infection test as gold standard for diagnosis and confirmation. The real-time RT-PCR was adapted with the goal to detect the whole spectrum of lyssavirus species, for nine of which synthesized DNA fragments were used. For the detection of species, seven probes were developed. Serial dilutions of the rabies virus strain CVS-11 showed a 100-fold higher sensitivity of real-time PCR compared to heminested RT-PCR. Using a panel of thirty-one lyssaviruses representing four species, the suitability of the protocol could be shown. Phylogenetic analysis of the sequences obtained by heminested PCR allowed correct classification of all viruses used. PMID:26464934

  15. Molecular identification of Salmonella Infantis isolated from backyard chickens and detection of their resistance genesby PCR

    PubMed Central

    Ghoddusi, A; Nayeri Fasaei, B; Karimi, V; Ashrafi Tamai, I; Moulana, Z; Zahraei Salehi, T

    2015-01-01

    This study aims at molecular identification of Salmonella Infantis isolated from backyard chickens and the detection of their antibiotic resistance genes. A total of 46 Salmonella-suspected samples isolated from backyard chickens of northern Iran were collected. Serotyping was done by the traditional method and then confirmed by PCR. Antimicrobial susceptibility of the isolates against 13 antimicrobial agents was determined by the standard disk diffusion method. There were 44 samples identified as Salmonella. Serotyping results showed that all 44 isolates belonged to serogroup C1 and serovar Infantis. The most resistance observed was to tetracycline and doxycycline (100%), chloramphenicol (79%) and florfenicol (72%). The floR, catI, tetA and tetG genes were used for the detection of florfenicol chloramphenicol and tetracycline resistance. In order to identify the phenotypic resistance in strains which showed resistance genes by PCR, colony PCR and culture on plates each containing antibiotic was performed simultaneously. All the Salmonella Infantis resistant to florfenicol and chloramphenicol harbored floR and catI. None of the Salmonella resistant to tetracycline carried tetA or tetG. The result of colony PCR and culture in antibiotic medium confirmed the results of PCR and indicated phenotypic resistance in these samples. PMID:27175192

  16. A multiplex PCR method of detecting recombinant DNAs from five lines of genetically modified maize.

    PubMed

    Matsuoka, T; Kuribara, H; Akiyama, H; Miura, H; Goda, Y; Kusakabe, Y; Isshiki, K; Toyoda, M; Hino, A

    2001-02-01

    Seven lines of genetically modified (GM) maize have been authorized in Japan as foods and feeds imported from the USA. We improved a multiplex PCR method described in the previous report in order to distinguish the five lines of GM maize. Genomic DNA was extracted from GM maize with a silica spin column kit, which could reduce experimental time and improve safety in the laboratory and potentially in the environment. We sequenced recombinant DNA (r-DNA) introduced into GM maize, and re-designed new primer pairs to increase the specificity of PCR to distinguish five lines of GM maize by multiplex PCR. A primer pair for the maize intrinsic zein gene (Ze1) was also designed to confirm the presence of amplifiable maize DNA. The lengths of PCR products using these six primer pairs were different. The Ze1 and the r-DNAs from the five lines of GM maize were qualitatively detected in one tube. The specific PCR bands were distinguishable from each other on the basis of the expected length. The r-DNA could be detected from maize samples containing 0.5% of each of the five lines of GM maize. The sensitivity would be acceptable to secure the verification of non-GMO materials and to monitor the reliability of the labeling system. PMID:11383153

  17. [PCR for detection and characterization of parasites (Leishmania, Echinococcus, Microsporodia, Giardia)].

    PubMed

    Mathis, A; Deplazes, P; Köhler, P; Eckert, J

    1996-01-01

    The application of PCR for the diagnosis and the characterisation of parasites is discussed using four examples. (1) A PCR assay that was developed for the detection of Leishmania was shown to be as highly sensitive as in vitro cultivation of the parasites from lymph node aspirates and bone marrow biopsies. (2) Single eggs of Echinococcus multilocularis can principally be detected and identified by PCR. However, a cumbersome sample preparation is inevitable in order to remove PCR-inhibitory substances present in fox faeces. (3) Analysis of the amplified SSU rRNA gene of Encephalitozoon-like isolates from humans, rabbits and farm foxes confirmed for the first time that E. cuniculi-isolates from humans and animals are indistinguishable and that they are of importance as opportunistic parasites in HIV-infected patients. (4) Swiss isolates of Giardia originating from humans, calves, sheep and a dog could be allocated into three distinct genetic groups by analysing several PCR-amplified genes that encode for variant-specific surface proteins. There was no evidence for the presence of host-specific genotypes. PMID:8721187

  18. Legionella detection by culture and qPCR: Comparing apples and oranges.

    PubMed

    Whiley, Harriet; Taylor, Michael

    2016-02-01

    Legionella spp. are the causative agent of Legionnaire's disease and an opportunistic pathogen of significant public health concern. Identification and quantification from environmental sources is crucial for identifying outbreak origins and providing sufficient information for risk assessment and disease prevention. Currently there are a range of methods for Legionella spp. quantification from environmental sources, but the two most widely used and accepted are culture and real-time polymerase chain reaction (qPCR). This paper provides a review of these two methods and outlines their advantages and limitations. Studies from the last 10 years which have concurrently used culture and qPCR to quantify Legionella spp. from environmental sources have been compiled. 26/28 studies detected Legionella at a higher rate using qPCR compared to culture, whilst only one study detected equivalent levels of Legionella spp. using both qPCR and culture. Aggregating the environmental samples from all 28 studies, 2856/3967 (72%) tested positive for the presence of Legionella spp. using qPCR and 1331/3967 (34%) using culture. The lack of correlation between methods highlights the need to develop an acceptable standardized method for quantification that is sufficient for risk assessment and management of this human pathogen. PMID:24580080

  19. Detection and identification of Malassezia species in domestic animals and aquatic birds by PCR-RFLP.

    PubMed

    Zia, M; Mirhendi, H; Toghyani, M

    2015-01-01

    The present study aimed at detection and species-level identification of the Malassezia yeasts in domestic animals and aquatic birds by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Samples were collected using tape strips and swabs from 471 animals including 97 horses, 102 cattle, 105 sheep, 20 camels, 60 dogs, 30 cats, 1 hamster, 1 squirrel, 50 aquatic birds and 5 turkeys. Tape-strip samples were examined by direct microscopy. All samples were inoculated on modified Leeming and Notman agar medium. DNA extracted from the yeast colonies was amplified by PCR using primers specific for 26S rDNA. RFLP of the PCR products was performed using Hin6I enzyme, and PCR and RFLP products were visualized by agarose gel electrophoresis. Malassezia yeasts were detected at the following frequencies: 15.46% in horses, 12.74% in cattle, 12.38% in sheep, 28.33% in dogs, 26.66% in cats and 26% in aquatic birds. Eighty colonies of 6 species were isolated: Malassezia globosa 41.25%, Malassezia furfur 22.5%, Malassezia restricta 15%, Malassezia sympodialis 15%, Malassezia pachydermatis 5% and Malassezia slooffiae 1.25%. Therefore different lipophilic Malassezia species are found in a wide diversity of animals and aquatic birds. PCR-RFLP is a suitable technique for identification of different Malassezia species. PMID:27175148

  20. Accuracy of universal polymerase chain reaction (PCR) for detection of bacterial meningitis among suspected patients

    PubMed Central

    Moayedi, Ali Reza; Nejatizadeh, Abdolazim; Mohammadian, Maryam; Rahmati, Mohammad Bagher; Namardizadeh, Vahideh

    2015-01-01

    Introduction Central nervous system (CNS) infections are life-threatening diseases caused by viral, bacterial, parasitic and fungal microorganisms. The aim of this study was to determine the accuracy of universal polymerase chain reaction (PCR) for the detection of bacterial meningitis among patients who were referred to Koodakan Hospital in Bandar Abbas because they were suspected of having the disease. Methods This study was conducted in 2013 on the patients who were admitted to Bandar Abbas’ Koodakan Hospital because they were suspected of having meningitis. A questionnaire, including demographic data, was completed for each patient. Universal PCR, Cerebrospinal fluid (CSF) analysis, and gram staining and cultures were done for all the patients. The data were analyzed using SPSS software. Results Among the 100 patients studied 59 (59%) were male and 41 (41%) were female. No patient in our study had a positive smear and culture for meningitis. Among the patients with negative smears and cultures six (6%) had positive universal PCR, and 94 (94%) had negative universal PCR. Based on these results, PCR had 95% specificity and 100% negative predictive value for the prediction of meningitis. In 30 patients (30%), the biochemical analysis of CSF were in favor of meningitis. Among the 30 patients, six patients (20%) had positive universal PCR and 24 patients (80%) had negative universal PCR. Conclusion Based on our results, the universal PCR test is useful in the diagnosis of bacterial meningitis in children. We recommend using it in combination with other tests, such as CSF analysis, for diagnosis of bacterial meningitis. PMID:26816587

  1. APPLICATION OF POLYMERASE CHAIN REACTION (PCR) AND PCR BASED RESTRICTION FRAGMENT LENGTH POLYMORPHISM FOR DETECTION AND IDENTIFICATION OF DERMATOPHYTES FROM DERMATOLOGICAL SPECIMENS

    PubMed Central

    Bagyalakshmi, R; Senthilvelan, B; Therese, K L; Murugusundram, S; Madhavan, H N

    2008-01-01

    Objective: To develop and optimize polymerase chain reaction-based restriction fragment length polymorphism (PCR-RFLP) targeting 18S rDNA and internal transcribed spacer (ITS) region of fungi for rapid detection and identification of dermatophytes. Materials and Methods: Two PCR-RFLP methods targeting 18S rDNA and ITS regions of fungi were optimized using standard and laboratory isolates of dermatophytes and other fungi. Sixty-eight dermatological clinical specimens (nail clippings (56), material obtained from blisters (8), hair root (2), scraping from scaly plaque of foot (1) and skin scraping (1) collected by the dermatologist were subjected to both the optimized PCR-RFLP and conventional mycological (smear and culture) methods. Results: PCRs targeting 18S rDNA and the ITS region were sensitive to detect 10 picograms and 1 femtogram of T. rubrum DNA, respectively. PCR targeting 18S rDNA was specific for dermatophytes and subsequent RFLP identified them to species level. PCR-RFLP targeting the ITS region differentiated dermatophytes from other fungi with identification to species level. Among the 68 clinical specimens tested, both PCR-RFLP methods revealed the presence of dermatophytes in 27 cases (39.7%), whereas culture revealed the same only in 2 cases (7.40%), increasing the clinical sensitivity by 32.3%. Among 20 smear positive specimens, both PCR-RFLP methods detected dermatophytes in 12 (17.6%). Both the methods detected the presence of dermatophytes in 13 (19.11%) smear and culture negative specimens, increasing the clinical sensitivity by 36.1%. Conclusion: PCR-RFLP methods targeting 18S rDNA and the ITS regions of fungi were specific and highly sensitive for detection and speciation of dermatophytes. PMID:19967012

  2. Evaluation of Five Real-Time PCR Assays for Detection of Mycoplasma pneumoniae

    PubMed Central

    Jacobs, Enno

    2014-01-01

    Four commercial real-time PCR assays to detect Mycoplasma pneumoniae were tested, and the results were compared with the results for an in-house approach. Despite differences of crossing threshold values of up to 4, assays were able to detect at least 20 CFU/5 μl (52 fg DNA/5 μl) of sample with the Diagenode kit showing the best clinical sensitivity. PMID:25210063

  3. Detection of Echinococcus multilocularis in faeces by nested PCR with the use of diluted DNA samples.

    PubMed

    Karamon, J

    2014-01-01

    The aim of this study was to choose the optimal variant of PCR examination of faeces to detect Echinococcus multilocularis infection which would allow to reduce the influence of different inhibitors in faeces. The investigation was carried out by comparison of 3 different methods of DNA isolation from faeces and different DNA dilutions used in PCR. Thirty five intestines of red foxes were used. Small intestines were examined by the sedimentation and counting technique (SCT). Faeces were collected from the rectum for PCR and flotation. DNA were isolated with the use of 3 different methods. Two methods were dedicated for faeces: method 1 (M1)--for larger samples and method 2 (M2) - for standard samples. The third method, method 3 (M3), was not dedicated for faeces. DNA samples were tested by nested PCR in 6 variants: not diluted (1/1) and 5 diluted (1/2.5, 1/5, 1/10. 1/20, 1/40). E. multilocularis was found by SCT in 18 from 35 (51.4%) intestines. Taenia-type eggs were detected only in 20.0% of faecal samples. In PCR the highest number of positive results (45.7%) were obtained during examination of DNA isolated by M1 method, and then 40.0% and 34.3%, respectively, for M2 and M3. In some samples positive results in PCR were obtained only in diluted DNA. For example, 8 from 12 positive samples isolated by M3 method gave the PCR negative results in non-diluted DNA and positive only after dilution 1:2.5, 1:10 or 1:20. Also 3 samples isolated by methods dedicated for stool gave positive results only after DNA dilution. The investigation has revealed that in copro-PCR for detection of E. multilocularis infection additional using of diluted DNA (besides non diluted) can avoid false negative results causing by PCR inhibition. In the best method of DNA isolation (M1), the use of non diluted DNA sample together with diluted in proportion 1:10 seems to be optimal. PMID:24724473

  4. 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

  5. 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

    2012-01-01

    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%. PMID:22843069

  6. A New Real-Time PCR Assay for Improved Detection of the Parasite Babesia microti

    PubMed Central

    Teal, Allen E.; Habura, Andrea; Ennis, Jill; Keithly, Janet S.

    2012-01-01

    Babesiosis is an emerging zoonosis with important public health implications, as the incidence of the disease has risen dramatically over the past decade. Because the current gold standard for detection of Babesia is microscopic examination of blood smears, accurate identification requires trained personnel. Species in the genus cannot be distinguished microscopically, and Babesia can also be confused with the early trophozoite stage (ring forms) of Plasmodium parasites. To allow more accurate diagnosis in a format that is accessible to a wider variety of laboratories, we developed a real-time PCR assay targeting the 18S rRNA gene of Babesia microti, the dominant babesiosis pathogen in the United States. The real-time PCR is performed on DNA extracted from whole-blood specimens and detects Babesia microti with a limit of detection of ∼100 gene copies in 5 μl of blood. The real-time PCR assay was shown to be 100% specific when tested against a panel of 24 organisms consisting of Babesia microti, other Babesia species, Plasmodium species, tick-borne and other pathogenic bacteria, and other blood-borne parasites. The results using clinical specimens show that the assay can detect infections of lower parasitemia than can be detected by microscopic examination. This method is therefore a rapid, sensitive, and accurate method for detection of Babesia microti in patient specimens. PMID:22170915

  7. Protocol: a highly sensitive RT-PCR method for detection and quantification of microRNAs

    PubMed Central

    Varkonyi-Gasic, Erika; Wu, Rongmei; Wood, Marion; Walton, Eric F; Hellens, Roger P

    2007-01-01

    MicroRNAs (miRNAs) are a class of small non-coding RNAs with a critical role in development and environmental responses. Efficient and reliable detection of miRNAs is an essential step towards understanding their roles in specific cells and tissues. However, gel-based assays currently used to detect miRNAs are very limited in terms of throughput, sensitivity and specificity. Here we provide protocols for detection and quantification of miRNAs by RT-PCR. We describe an end-point and real-time looped RT-PCR procedure and demonstrate detection of miRNAs from as little as 20 pg of plant tissue total RNA and from total RNA isolated from as little as 0.1 μl of phloem sap. In addition, we have developed an alternative real-time PCR assay that can further improve specificity when detecting low abundant miRNAs. Using this assay, we have demonstrated that miRNAs are differentially expressed in the phloem sap and the surrounding vascular tissue. This method enables fast, sensitive and specific miRNA expression profiling and is suitable for facilitation of high-throughput detection and quantification of miRNA expression. PMID:17931426

  8. Ultra-rapid real-time PCR for the detection of Paenibacillus larvae, the causative agent of American Foulbrood (AFB).

    PubMed

    Han, Sang-Hoon; Lee, Do-Bu; Lee, Dong-Woo; Kim, Eul-Hwan; Yoon, Byoung-Su

    2008-09-01

    A novel micro-PCR-based detection method, termed ultra-rapid real-time PCR, was applied to the development of a rapid detection for Paenibacillus larvae (P. larvae) which is the causative agent of American Foulbrood (AFB). This method was designed to detect the 16S rRNA gene of P. larvae with a micro-scale chip-based real-time PCR system, GenSpector TMC-1000, which has uncommonly fast heating and cooling rates (10 degrees C per second) and small reaction volume (6microl). In the application of ultra-rapid real-time PCR detection to an AFB-infected larva, the minimum detection time was 7 min and 54s total reaction time (30 cycles), including the melting temperature analysis. To the best of our knowledge, this novel detection method is one of the most rapid real-time PCR-based detection tools. PMID:18571197

  9. PCR detection, characterization, and distribution of virulence genes in Aeromonas spp.

    PubMed

    Kingombe, C I; Huys, G; Tonolla, M; Albert, M J; Swings, J; Peduzzi, R; Jemmi, T

    1999-12-01

    We found 73.1 to 96.9% similarity by aligning the cytolytic enterotoxin gene of Aeromonas hydrophila SSU (AHCYTOEN; GenBank accession no. M84709) against aerolysin genes of Aeromonas spp., suggesting the possibility of selecting common primers. Identities of 90 to 100% were found among the eight selected primers from those genes. Amplicons obtained from Aeromonas sp. reference strains by using specific primers for each gene or a cocktail of primers were 232 bp long. Of hybridization group 4/5A/5B (HG4/5A/5B), HG9, and HG12 or non-Aeromonas reference strains, none were positive. PCR-restriction fragment length polymorphism (PCR-RFLP) with HpaII yielded three types of patterns. PCR-RFLP 1 contained two fragments (66 and 166 bp) found in HG6, HG7, HG8, HG10, and HG11. PCR-RFLP 2 contained three fragments (18, 66, and 148 bp) found in HG1, HG2, HG3, and HG11. PCR-RFLP 3, with four fragments (7, 20, 66, and 139 bp), was observed only in HG13. PCR-amplicon sequence analysis (PCR-ASA) revealed three main types. PCR-ASA 1 had 76 to 78% homology with AHCYTOEN and included strains in HG6, HG7, HG8, HG10, and HG11. PCR-ASA 2, with 82% homology, was found only in HG13. PCR-ASA 3, with 91 to 99% homology, contained the strains in HG1, HG2, HG3, and HG11. This method indicated that 37 (61%) of the 61 reference strains were positive with the primer cocktail master mixture, and 34 (58%) of 59 environmental isolates, 93 (66%) of 141 food isolates, and 100 (67%) of 150 clinical isolates from around the world carried a virulence factor when primers AHCF1 and AHCR1 were used. In conclusion, this PCR-based method is rapid, sensitive, and specific for the detection of virulence factors of Aeromonas spp. It overcomes the handicap of time-consuming biochemical and other DNA-based methods. PMID:10583979

  10. [Detection and Serotyping of Streptococcus pneumoniae Carried in Healthy Adults with a Modified PCR Method].

    PubMed

    Ishihara, Yuka; Okamoto, Akira; Ohta, Michio

    2015-05-01

    Detection of Streptococcus pneumoniae colonized in the pharynx of healthy carriers currently relies on conventional culture methods of direct plating with pharyngeal swab specimens. The accurate measurement of the carriage of pneumococci, however, has not been necessarily achieved with these methods due to low density colonization and contamination of numerous oral streptococci that express α-hemolysis. A PCR-based detection method of pneumococci-specific for lytA as well as PCR serotyping of S. pneumoniae was recently developed and their effectiveness was confirmed. We modified the reaction conditions of these methods to improve the detection rate and applied them to the measurement of S. pneumoniae carried in healthy adults. Pharyngeal swab specimens obtained from 110 healthy volunteers over 40 and living in Nagoya were enriched for 5 hours with broth medium supplemented with rabbit serum and the template DNA for PCR was extracted from the mixed enriched culture. Of 110 specimens 36 (32.7%) were lytA-positive, the rate of which was much higher than the results of previous culture-based studies. The DNA template preparations were then used for PCR-based serotyping with primers specific for each of the types included in pneumococcal 23 valent vaccine (PPV23). We found that 28 out of 36 lytA-positive carriers were identified as being positive for the serotypes belonging to PPV23, although serotypes 6A and 6B were indistinguishable with the PCR method. The most frequent serotype was serotype 14, and serotypes 4, 18C, and 6A/B were also frequently identified. Five lytA-positive carriers were previously vaccinated with PPV23, and among them, 4 were positive for serotypes contained in PPV23. We recommend PCR-based identification and serotyping of S. pneumoniae in broth enrichment culture of pharyngeal swab specimens as a reliable method for the surveillance of healthy carriers with low density colonization. PMID:26552129

  11. Semiquantitative Multiplexed Tandem PCR for Detection and Differentiation of Four Theileria orientalis Genotypes in Cattle

    PubMed Central

    Perera, Piyumali K.; Gasser, Robin B.; Firestone, Simon M.; Smith, Lee; Roeber, Florian

    2014-01-01

    Oriental theileriosis is an emerging, tick-borne disease of bovines in the Asia-Pacific region and is caused by one or more genotypes of the Theileria orientalis complex. This study aimed to establish and validate a multiplexed tandem PCR (MT-PCR) assay using three distinct markers (major piroplasm surface protein, 23-kDa piroplasm membrane protein, and the first internal transcribed spacer of nuclear DNA), for the simultaneous detection and semiquantification of four genotypes (Buffeli, Chitose, Ikeda, and type 5) of the T. orientalis complex. Analytical specificity, analytical sensitivity, and repeatability of the established MT-PCR assay were assessed in a series of experiments. Subsequently, the assay was evaluated using 200 genomic DNA samples collected from cattle from farms on which oriental theileriosis outbreaks had occurred, and 110 samples from a region where no outbreaks had been reported. The results showed the MT-PCR assay specifically and reproducibly detected the expected genotypes (i.e., genotypes Buffeli, Chitose, Ikeda, and type 5) of the T. orientalis complex, reliably differentiated them, and was able to detect as little as 1 fg of genomic DNA from each genotype. The diagnostic specificity and sensitivity of the MT-PCR were estimated at 94.0% and 98.8%, respectively. The MT-PCR assay established here is a practical and effective diagnostic tool for the four main genotypes of T. orientalis complex in Australia and should assist studies of the epidemiology and pathophysiology of oriental theileriosis in the Asia-Pacific region. PMID:25339402

  12. Detection of toxigenic strains of Aeromonas species in foods by a multiplex PCR assay.

    PubMed

    Balakrishna, K; Murali, H S; Batra, H V

    2010-06-01

    Aeromonas hydrophila and other aeromonads are ubiquitous organisms found in meat, vegetables, drinking water and various other food items. They cause diarrhea and extra-intestinal infections in normal and immunocompromised patients. The aim of the study was to develop a multiplex PCR assay for the detection of virulence-associated genes of Aeromonas including hemolysin (hlyA), aerolysin (aerA), glycerophospholipid-cholesterol acyl transferase (GCAT) alongwith a 16S rRNA gene. Internal amplification control (IAC), which was coamplified with aerA primers, was also included in this study. The results showed that all cultures of Aeromonas were accurately identified by the assay without showing non-specificity. A. hydrophila could be detected at a range of 10-50 CFU ml(-1) from experimentally spiked fish, chicken and milk samples following overnight enrichment in alkaline peptone water supplemented with 10 μg/ml ampicillin (APW-A) by this multiplex PCR (mPCR). When evaluated on a total of 74 naturally occurring food samples, four samples were identified to contain Aeromonas by mPCR. All these results were compared to the conventional culture, isolation and biochemical identification procedures. The high throughput and cost-effective mPCR method developed in this study could provide a powerful tool for detection of pathogenic Aeromonas spp. from food and environmental samples and in addition, the method has advantages in terms of specificity, sensitivity and ease of use compared to other reported PCR methods and DNA hybridization assays. PMID:23100820

  13. 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. PMID:24006008

  14. Development and Evaluation of a Novel Real-Time PCR for Pan-Dermatophyte Detection in Nail Specimens.

    PubMed

    Gong, Jie; Ran, Menglong; Wang, Xiaowen; Wan, Zhe; Li, Ruoyu

    2016-02-01

    An accurate diagnosis of tinea unguium is necessary for the selection of antimycotics and successful treatment. To rapidly and accurately identify the aetiological agents causing tinea unguium, we improved upon the conventional boiling method for DNA extraction and developed a novel real-time PCR detection system that includes two assays. The two assays, based on the amplification of ribosomal internal transcribed spacer regions and 28S rDNA, were designed to detect pan-dermatophyte and Trichophyton rubrum, respectively. The analytical sensitivities of both assays permitted the detection of ten copies of plasmid DNA template. The analytical specificity of the detection system was confirmed using 11 dermatophyte strains and 25 non-dermatophyte strains. In total, 165 nail specimens were examined by microscopy, culture, conventional PCR, and the novel real-time PCR method. Real-time PCR gave positive results in 47.3 % of the specimens (78), a rate exceeding those obtained using microscopy (72, 43.6 %), conventional PCR (69, 41.8 %), and culture (49, 29.7 %). All conventional PCR-positive specimens were detected by real-time PCR, and real-time PCR detected nine specimens that were missed by conventional PCR. The results from latent class analysis, and further calculations, showed that real-time PCR was the most sensitive method, but the diagnostic specificity of the four approaches was equivalent. In particular, molecular approaches may be more effective than microscopy and culture when the clinical symptoms of tinea unguium are not evident. PMID:26412381

  15. A rapid method for detecting specific amplified PCR fragments in microtiter plates.

    PubMed Central

    Ortiz, A; Ritter, E

    1996-01-01

    A simple method is presented to circumvent laborious and time consuming electrophoretic separations of specific PCR amplification products. Specific target DNA is amplified using nucleotides labelled with DIG-dUTP or biotin-dCTP. The labelled PCR products are separated from unincorporated nucleotides or oligonucleotides by using a positively charged DEAE cellulose matrix. Amplification products are visualized directly in the matrix using immunoenzymatic methods or streptavidin-conjugated enzymes. The detection process can be carried out within 2 h, allows the processing of large sample sizes and can potentially be automated. PMID:8774915

  16. Detection of Mycobacterium bovis DNA in nasal swabs from tuberculous cattle by a multiplex PCR

    PubMed Central

    de Souza Figueiredo, Eduardo Eustáquio; Carvalho, Ricardo Cezar Tavares; Silvestre, Flávia Galindo; Lilenbaum, Walter; Fonseca, Leila Sousa; Silva, Joab Trajano; Paschoalin, Vânia Margaret Flosi

    2010-01-01

    Detection of tuberculosis in cattle relies on the intradermal tuberculin test (ITT), but a definitive diagnosis requires identification of the pathogen after the animal is slaughtered. DNA in nasal swabs from 50 cows was analyzed by m-PCR, targeting for the RvD1-Rv2031c and IS6110 sequences. M. bovis was identified in two of 34 tuberculous cows (5.9%). The use of mPCR of nasal swabs as an in vivo diagnostic tool for bovine tuberculosis is suggested. PMID:24031509

  17. European validation of Real-Time PCR method for detection of Salmonella spp. in pork meat.

    PubMed

    Delibato, Elisabetta; Rodriguez-Lazaro, David; Gianfranceschi, Monica; De Cesare, Alessandra; Comin, Damiano; Gattuso, Antonietta; Hernandez, Marta; Sonnessa, Michele; Pasquali, Frédérique; Sreter-Lancz, Zuzsanna; Saiz-Abajo, María-José; Pérez-De-Juan, Javier; Butrón, Javier; Prukner-Radovcic, Estella; Horvatek Tomic, Danijela; Johannessen, Gro S; Jakočiūnė, Džiuginta; Olsen, John E; Chemaly, Marianne; Le Gall, Francoise; González-García, Patricia; Lettini, Antonia Anna; Lukac, Maja; Quesne, Segolénè; Zampieron, Claudia; De Santis, Paola; Lovari, Sarah; Bertasi, Barbara; Pavoni, Enrico; Proroga, Yolande T R; Capuano, Federico; Manfreda, Gerardo; De Medici, Dario

    2014-08-01

    The classical microbiological method for detection of Salmonella spp. requires more than five days for final confirmation, and consequently there is a need for an alternative methodology for detection of this pathogen particularly in those food categories with a short shelf-life. This study presents an international (at European level) ISO 16140-based validation study of a non-proprietary Real-Time PCR-based method that can generate final results the day following sample analysis. It is based on an ISO compatible enrichment coupled to an easy and inexpensive DNA extraction and a consolidated Real-Time PCR assay. Thirteen laboratories from seven European Countries participated to this trial, and pork meat was selected as food model. The limit of detection observed was down to 10 CFU per 25 g of sample, showing excellent concordance and accordance values between samples and laboratories (100%). In addition, excellent values were obtained for relative accuracy, specificity and sensitivity (100%) when the results obtained for the Real-Time PCR-based methods were compared to those of the ISO 6579:2002 standard method. The results of this international trial demonstrate that the evaluated Real-Time PCR-based method represents an excellent alternative to the ISO standard. In fact, it shows an equal and solid performance as well as it reduces dramatically the extent of the analytical process, and can be easily implemented routinely by the Competent Authorities and Food Industry laboratories. PMID:24513055

  18. Detection by real time PCR of walnut allergen coding sequences in processed foods.

    PubMed

    Linacero, Rosario; Ballesteros, Isabel; Sanchiz, Africa; Prieto, Nuria; Iniesto, Elisa; Martinez, Yolanda; Pedrosa, Mercedes M; Muzquiz, Mercedes; Cabanillas, Beatriz; Rovira, Mercè; Burbano, Carmen; Cuadrado, Carmen

    2016-07-01

    A quantitative real-time PCR (RT-PCR) method, employing novel primer sets designed on Jug r 1, Jug r 3, and Jug r 4 allergen-coding sequences, was set up and validated. Its specificity, sensitivity, and applicability were evaluated. The DNA extraction method based on CTAB-phenol-chloroform was best for walnut. RT-PCR allowed a specific and accurate amplification of allergen sequence, and the limit of detection was 2.5pg of walnut DNA. The method sensitivity and robustness were confirmed with spiked samples, and Jug r 3 primers detected up to 100mg/kg of raw walnut (LOD 0.01%, LOQ 0.05%). Thermal treatment combined with pressure (autoclaving) reduced yield and amplification (integrity and quality) of walnut DNA. High hydrostatic pressure (HHP) did not produce any effect on the walnut DNA amplification. This RT-PCR method showed greater sensitivity and reliability in the detection of walnut traces in commercial foodstuffs compared with ELISA assays. PMID:26920302

  19. Development of a quantitative PCR for the detection of Rangelia vitalii.

    PubMed

    Paim, Francine Chimelo; dos Santos, Andrea Pires; do Nascimento, Naíla Cannes; Lasta, Camila Serina; Oliveira, Simone Tostes; Messick, Joanne Belle; Lopes, Sonia Terezinha dos Anjos

    2016-02-15

    The aim of this study was to develop and validate a SYBR Green qPCR assay to detect and quantify a fragment of the 18S rRNA gene of Rangelia vitalii in canine blood. Repeatability of the qPCR was determined by the intra- and inter-assay variations. The qPCR showed efficiency of E=101.30 (r(2)=0.996), detecting as few as one copy of plasmid containing the target DNA. Specificity of the assay was performed using DNA samples of Babesia canis, B. gibsoni, Ehrlichia canis, E. ewingii and Leishmania sp. No cross-reactivity was observed. Field samples consisting of blood from 265 dogs from Porto Alegre, Brazil were also tested. A total of 24 (9.05%) samples were positive for R. vitalii. Amplicons of 50% of positive samples were confirmed to be R. vitalii by Sanger sequencing. The positive samples had an average of 3.5×10(5) organisms/mL of blood (range: 1.27×10(3)-1.88×10(6)) based on the plasmid-generated standard curve. In conclusion, the SYBR Green qPCR assay developed herein is sensitive and specific and can be used as a diagnostic tool for detection and quantification of R. vitalii in canine blood samples. PMID:26827871

  20. Detection of Zika virus by SYBR green one-step real-time RT-PCR.

    PubMed

    Xu, Ming-Yue; Liu, Si-Qing; Deng, Cheng-Lin; Zhang, Qiu-Yan; Zhang, Bo

    2016-10-01

    The ongoing Zika virus (ZIKV) outbreak has rapidly spread to new areas of Americas, which were the first transmissions outside its traditional endemic areas in Africa and Asia. Due to the link with newborn defects and neurological disorder, numerous infected cases throughout the world and various mosquito vectors, the virus has been considered to be an international public health emergency. In the present study, we developed a SYBR Green based one-step real-time RT-PCR assay for rapid detection of ZIKV. Our results revealed that the real-time assay is highly specific and sensitive in detection of ZIKV in cell samples. Importantly, the replication of ZIKV at different time points in infected cells could be rapidly monitored by the real-time RT-PCR assay. Specifically, the real-time RT-PCR showed acceptable performance in measurement of infectious ZIKV RNA. This assay could detect ZIKV at a titer as low as 1PFU/mL. The real-time RT-PCR assay could be a useful tool for further virology surveillance and diagnosis of ZIKV. PMID:27444120

  1. Detection of Legionella pneumophila by PCR-ELISA method in industrial cooling tower water.

    PubMed

    Soheili, Majid; Nejadmoghaddam, Mohammad Reza; Babashamsi, Mohammad; Ghasemi, Jamileh; Jeddi Tehrani, Mahmood

    2007-11-15

    Water supply and Cooling Tower Water (CTW) are among the most common sources of Legionella pneumophila (LP) contamination. A nonradio active method is described to detect LP in industrial CTW samples. DNA was purified and amplified by nested -PCR with amplimers specific for the 16s rRNA gene of LP. The 5' end biotinylated oligomer probe was immobilized on sterptavidin B coated microtiter plates. The nested-PCR product was labeled with digoxigenin and then hybridized with 5'-biotinylated probes. The amplification products were detected by using proxidase-labled anti dioxygenin antibody in a colorimetric reaction. The assay detected LP present in 1 L of 5 CTW samples examined. All of the samples were Legionella positive in both culture and PCR-ELISA methods. The PCR-ELISA assay appears to exhibit high specificity and is a more rapid technique in comparison with bacterial culture method. Thus could prove suitable for use in the routine examination of industrial CTW contamination. PMID:19090273

  2. PCR-based detection of gene transfer vectors: application to gene doping surveillance.

    PubMed

    Perez, Irene C; Le Guiner, Caroline; Ni, Weiyi; Lyles, Jennifer; Moullier, Philippe; Snyder, Richard O

    2013-12-01

    Athletes who illicitly use drugs to enhance their athletic performance are at risk of being banned from sports competitions. Consequently, some athletes may seek new doping methods that they expect to be capable of circumventing detection. With advances in gene transfer vector design and therapeutic gene transfer, and demonstrations of safety and therapeutic benefit in humans, there is an increased probability of the pursuit of gene doping by athletes. In anticipation of the potential for gene doping, assays have been established to directly detect complementary DNA of genes that are top candidates for use in doping, as well as vector control elements. The development of molecular assays that are capable of exposing gene doping in sports can serve as a deterrent and may also identify athletes who have illicitly used gene transfer for performance enhancement. PCR-based methods to detect foreign DNA with high reliability, sensitivity, and specificity include TaqMan real-time PCR, nested PCR, and internal threshold control PCR. PMID:23912835

  3. RT-PCR is a more accurate diagnostic tool for detection of BCR-ABL rearrangement

    SciTech Connect

    Zehnbauer, B.A.; Allen, A.P.; McGrath, S.D.

    1994-09-01

    Detection of the Philadelphia chromosome (Ph1) or genomic Southern hybridization for clonal gene rearrangement (GSH-R) has provided very specific identification of BCR-ABL gene rearrangement. Reverse transcriptase-polymerase chain reaction (RT-PCR) is diagnostic for patterns of BCR-ABL expression which are undetected by GSH-R and/or Ph1 and provides increased sensitivity both at diagnosis and in detection of minimal residual leukemia. Fifty-three specimens (of 150 tested from 119 consecutive leukemia patients) were RT-PCR positive for BCR-ABL gene expression confirmed by hybridization of PCR products with b{sub 3}a{sub 2}, b{sub 2}a{sub 2}, or e{sub 1}a{sub 2} junction-specific oligonucleotides. In 6 cases of CML with GSH-R{sup {minus}}at diagnosis, RT-PCR provided specific BCR-ABL identification. Deletion of BCR regions, low mitotic index, or e{sub 1}a{sub 2} expression caused failure to detect GSH-R or Ph1 translocation.

  4. Interlaboratory Validation for a Real-Time PCR Salmonella Detection Method Using the ABI 7500 FAST Real-Time PCR System.

    PubMed

    Cheng, Chorng-Ming; Doran, Tara; Lin, Wen; Chen, Kai-Shun; Williams-Hill, Donna; Pamboukian, Ruiqing

    2015-06-01

    Sixteen FERN (Food Emergency Response Network) member laboratories collaborated in this study to verify extension of the real-time PCR Salmonella detection method originally designed for the single-tube Cepheid SmartCycler II and validated against the Salmonella method of the U. S. Food and Drug Administration Bacteriological Analytical Manual to the Applied Biosystems (ABI) 7500 FAST Real-Time PCR system multiwell plate platform. Four foods were selected for this study: chili powder, soft cheese, fish, and tomatoes; these foods represent products that are commonly analyzed for the presence of Salmonella for regulatory purposes. Each food consisted of six uninoculated control samples, six samples inoculated with low Salmonella levels (target 1 to 5 CFU/25 g), and six samples inoculated with high levels (target 10 to 50 CFU/25 g). All samples were tested for Salmonella using the 24-h quantitative PCR (qPCR) method for detecting Salmonella, which utilizes modified buffered peptone water as the sole enrichment medium and an internal control for the qPCR. Each of these 18 samples was individually analyzed for Salmonella by the collaborating laboratories using both the ABI 7500 FAST system (alternative method) and the SmartCycler II system (reference method). Statistical analysis of the data revealed no significant difference (P ≥ 0.05) between these two qPCR platforms except for the chili powder samples. The differences noted with chili powder (P = 0.0455) were attributed to the enhanced sensitivity of the ABI 7500 FAST system compared with the SmartCycler II system. The detection limit of both qPCR methods was 0.02 to 0.15 CFU/g. These results provide a solid basis for extending the 24-h qPCR Salmonella method to the ABI 7500 FAST system for high-throughput detection of Salmonella in foods. PMID:26038901

  5. Use of TaqMan® real-time PCR for rapid detection of Salmonella enterica serovar Typhi.

    PubMed

    Ranjbar, Reza; Naghoni, Ali; Farshad, Shohreh; Lashini, Hadi; Najafi, Ali; Sadeghifard, Nourkhoda; Mammina, Caterina

    2014-06-01

    We evaluated the performances of a newly designed real-time polymerase chain reaction (PCR) assay using TaqMan® probes to detect Salmonella Typhi. TaqMan® real-time PCR assays were performed by designed primers and probe based on the staG gene for detecting S. Typhi. The specificity of the assay was evaluated on 15 Salmonella serovars. The analytical specificity was evaluated on 20 non-Salmonella microorganisms. The analytical sensitivity was assessed using decreasing DNA quantities of S. Typhi ATCC 19430. Finally the detection capability of the TaqMan® real-time PCR assay on isolates recovered from patients with Salmonella infections was compared to the conventional PCR assay. Only S. Typhi strain had positive results when subjected to the assay using Typhi-specific real-time PCR. No amplification products were observed in real-time PCR with any of the non-Salmonella microorganisms tested. The TaqMan® real-time PCR was more sensitive than the conventional PCR. In conclusion, we found that the easy-to-use real-time PCR assays were faster than conventional PCR systems. The staG-based TaqMan® real-time PCR assay showed to be specific and sensitive method for the safe and rapid detection of the S. Typhi. PMID:24939681

  6. Detection of Infectious Adenovirus in Cell Culture by mRNA Reverse Transcription-PCR

    PubMed Central

    Ko, Gwangpyo; Cromeans, Theresa L.; Sobsey, Mark D.

    2003-01-01

    We have developed and evaluated the reverse transcription (RT)-PCR detection of mRNA in cell culture to assay infectious adenoviruses (Ads) by using Ad type 2 (Ad2) and Ad41 as models. Only infectious Ads are detected because they are the only ones able to produce mRNA during replication in cell culture. Three primer sets for RT-PCR amplification of mRNA were evaluated for their sensitivity and specificity: a conserved region of late mRNA transcript encoding a virion structural hexon protein and detecting a wide range of human Ads and two primer sets targeting a region of an early mRNA transcript that specifically detects either Ad2 and Ad5 or Ad40 and Ad41. The mRNAs of infected A549 and Graham 293 cells were recovered from cell lysates with oligo(dT) at different time periods after infection and treated with RNase-free DNase to remove residual contaminating DNA, and then Ad mRNA was detected by RT-PCR assay. The mRNA of Ad2 was detected as early as 6 h after infection at 106 infectious units (IU) per cell culture and after longer incubation times at levels as low as 1 to 2 IU per cell culture. The mRNA of Ad41 was detected as soon as 24 h after infection at 106 IU per cell culture and at levels as low as 5 IU per cell culture after longer incubation times. To confirm the detection of only infectious viruses, it was shown that no mRNA was detected from Ad2 and Ad41 inactivated by free chlorine or high doses of collimated, monochromatic (254-nm) UV radiation. Detection of Ad2 mRNA exactly coincided with the presence of virus infectivity detected by cytopathogenic effects in cell cultures, but mRNA detection occurred sooner. These results suggest that mRNA detection by RT-PCR assay in inoculated cell cultures is a very sensitive, specific, and rapid method by which to detect infectious Ads in water and other environmental samples. PMID:14660388

  7. The use of singleplex and nested PCR to detect Batrachochytrium dendrobatidis in free-living frogs

    PubMed Central

    Coutinho, Selene Dall'Acqua; Burke, Julieta Catarina; de Paula, Catia Dejuste; Rodrigues, Miguel Trefaut; Catão-Dias, José Luiz

    2015-01-01

    Many microorganisms are able to cause diseases in amphibians, and in the past few years one of the most reported has been Batrachochytrium dendrobatidis. This fungus was first reported in Brazil in 2005; following this, other reports were made in specimens deposited in museum collections, captive and free-living frogs. The aim of this study was to compare singleplex and nested-PCR techniques to detect B. dendrobatidis in free-living and apparently healthy adult frogs from the Brazilian Atlantic Forest. The sample collection area was a protected government park, with no general entrance permitted and no management of the animals there. Swabs were taken from the skin of 107 animals without macroscopic lesions and they were maintained in ethanol p.a. Fungal DNA was extracted and identification of B. dendrobatidis was performed using singleplex and nested-PCR techniques, employing specific primers sequences. B. dendrobatidis was detected in 61/107 (57%) and 18/107 (17%) animals, respectively by nested and singleplex-PCR. Nested-PCR was statistically more sensible than the conventional for the detection of B. dendrobatidis (Chi-square = 37.1; α = 1%) and the agreement between both techniques was considered just fair (Kappa = 0.27). The high prevalence obtained confirms that these fungi occur in free-living frogs from the Brazilian Atlantic Forest with no macroscopic lesions, characterizing the state of asymptomatic carrier. We concluded that the nested-PCR technique, due to its ease of execution and reproducibility, can be recommended as one of the alternatives in epidemiological surveys to detect B. dendrobatidis in healthy free-living frog populations. PMID:26273273

  8. The use of singleplex and nested PCR to detect Batrachochytrium dendrobatidis in free-living frogs.

    PubMed

    Coutinho, Selene Dall'Acqua; Burke, Julieta Catarina; de Paula, Catia Dejuste; Rodrigues, Miguel Trefaut; Catão-Dias, José Luiz

    2015-06-01

    Many microorganisms are able to cause diseases in amphibians, and in the past few years one of the most reported has been Batrachochytrium dendrobatidis. This fungus was first reported in Brazil in 2005; following this, other reports were made in specimens deposited in museum collections, captive and free-living frogs. The aim of this study was to compare singleplex and nested-PCR techniques to detect B. dendrobatidis in free-living and apparently healthy adult frogs from the Brazilian Atlantic Forest. The sample collection area was a protected government park, with no general entrance permitted and no management of the animals there. Swabs were taken from the skin of 107 animals without macroscopic lesions and they were maintained in ethanol p.a. Fungal DNA was extracted and identification of B. dendrobatidis was performed using singleplex and nested-PCR techniques, employing specific primers sequences. B. dendrobatidis was detected in 61/107 (57%) and 18/107 (17%) animals, respectively by nested and singleplex-PCR. Nested-PCR was statistically more sensible than the conventional for the detection of B. dendrobatidis (Chi-square = 37.1; α = 1%) and the agreement between both techniques was considered just fair (Kappa = 0.27). The high prevalence obtained confirms that these fungi occur in free-living frogs from the Brazilian Atlantic Forest with no macroscopic lesions, characterizing the state of asymptomatic carrier. We concluded that the nested-PCR technique, due to its ease of execution and reproducibility, can be recommended as one of the alternatives in epidemiological surveys to detect B. dendrobatidis in healthy free-living frog populations. PMID:26273273

  9. Comparison of Stool Antigen Detection Kits to PCR for Diagnosis of Amebiasis ▿

    PubMed Central

    Stark, D.; van Hal, S.; Fotedar, R.; Butcher, A.; Marriott, D.; Ellis, J.; Harkness, J.

    2008-01-01

    The present study was conducted to compare two stool antigen detection kits with PCR for the diagnosis of Entamoeba histolytica infections by using fecal specimens submitted to the Department of Microbiology at St. Vincent's Hospital, Sydney, and the Institute of Medical and Veterinary Science, Adelaide, Australia. A total of 279 stool samples containing the E complex (E. histolytica, Entamoeba dispar, and Entamoeba moshkovskii) were included in this study. The stool specimens were tested by using two commercially produced enzyme immunoassays (the Entamoeba CELISA PATH and TechLab E. histolytica II kits) to detect antigens of E. histolytica. DNA was extracted from all of the samples with a Qiagen DNA stool mini kit (Qiagen, Hilden, Germany), and a PCR targeting the small-subunit ribosomal DNA was performed on all of the samples. When PCR was used as a reference standard, the CELISA PATH kit showed 28% sensitivity and 100% specificity. The TechLab ELISA (enzyme-linked immunosorbent assay) kit did not prove to be useful in detecting E. histolytica, as it failed to identify any of the E. histolytica samples which were positive by PCR. With the TechLab kit, cross-reactivity was observed for three specimens, one of which was positive for both E. dispar and E. moshkovskii while the other two samples contained E. moshkovskii. Quantitative assessment of the PCR and ELISA results obtained showed that the ELISA kits were 1,000 to 10,000 times less sensitive, and our results show that the CELISA PATH kit and the TechLab ELISA are not useful for the detection of E. histolytica in stool samples from patients in geographical regions where this parasite is not endemic. PMID:18367563

  10. Development of PCR-based methods for detection of Sphaerothecum destruens in fish tissues.

    PubMed

    Mendonca, Holly L; Arkush, Kristen D

    2004-11-01

    Single-round and nested polymerase chain reaction (PCR) tests were developed for amplification of a 434 bp fragment of the small subunit ribosomal RNA (18S rRNA) gene from Sphaerothecum destruens, previously known as the rosette agent, an intracellular parasite of salmonid fishes. Both tests have successfully amplified S. destruens-specific DNA from different isolates of S. destruens but not from related organisms. The limits of detection using the nested PCR test were 1 pg for purified S. destruens genomic DNA and 0.1 fg for plasmid DNA. We conducted 2 experimental transmission studies, consisting of injection or waterborne exposure of juvenile winter-run Chinook salmon Oncorhynchus tshawytscha to spore stages of the parasite. In the injection study, parasite DNA was detected in 100% of kidney samples from exposed fish (n = 83) at 1 and 3 mo post-exposure using nested PCR, versus 98% using microscopic analysis of Gram-stained impression smears made from the kidney. Following waterborne exposure, fish were sampled over the course of a year. From each fish, samples of gill, liver, posterior intestine and kidney were analyzed. S. destruens-specific DNA was detected most often in gill and kidney over the course of the experiment, and 71% (64/90) of the exposed fish were identified as positive for S. destruens using the nested PCR test, versus 16% (14/90) using microscopic analysis of Gram-stained kidney smears. Natural infections in captive broodstock of adult winter-run Chinook salmon, originally diagnosed by examination of Gram-stained kidney smears, were confirmed using the nested PCR test in all fish examined (15/15). Further, the nested test amplified parasite-specific DNA from other tissues in these fish with varying frequencies. This report introduces the first DNA-based detection method for S. destruens, to be used alone as a diagnostic tool or in conjunction with histologic tests for confirmatory identification of the parasite. PMID:15609874

  11. Sensitive PCR Detection of Meloidogyne arenaria, M. incognita, and M. javanica Extracted from Soil

    PubMed Central

    Qiu, Jinya Jack; Westerdahl, Becky B.; Anderson, Cindy; Williamson, Valerie M.

    2006-01-01

    We have developed a simple PCR assay protocol for detection of the root-knot nematode (RKN) species Meloidogyne arenaria, M. incognita, and M. javanica extracted from soil. Nematodes are extracted from soil using Baermann funnels and centrifugal flotation. The nematode-containing fraction is then digested with proteinase K, and a PCR assay is carried out with primers specific for this group of RKN and with universal primers spanning the ITS of rRNA genes. The presence of RKN J2 can be detected among large numbers of other plant-parasitic and free-living nematodes. The procedure was tested with several soil types and crops from different locations and was found to be sensitive and accurate. Analysis of unknowns and spiked soil samples indicated that detection sensitivity was the same as or higher than by microscopic examination. PMID:19259460

  12. A PCR method of detecting American Foulbrood (Paenibacillus larvae) in winter beehive wax debris.

    PubMed

    Ryba, Stepan; Titera, Dalibor; Haklova, Marcela; Stopka, Pavel

    2009-10-20

    The objective of this work was to create a fast and sensitive method of detecting Paenibacillus larvae from beehive debris based on PCR that does not require long-lasting cultivation steps. Various methods of extracting spores from beehive debris were compared: the original method of extraction of spores into toluene, and alternative spore extraction methods into Tween 80, into water, into isopropanol and into 95% ethanol. Isolation of DNA from various spore extractions was evaluated too. Best results were provided by isolation of DNA using the QIAamp DNA Mini Kit, without heat treatment. DNA of spores was detected by PCR from 0.25 g of beeswax debris, with the detected titer of 10(5) in 1g according to the cultivation tests. PMID:19559547

  13. Detection of almond allergen coding sequences in processed foods by real time PCR.

    PubMed

    Prieto, Nuria; Iniesto, Elisa; Burbano, Carmen; Cabanillas, Beatriz; Pedrosa, Mercedes M; Rovira, Mercè; Rodríguez, Julia; Muzquiz, Mercedes; Crespo, Jesus F; Cuadrado, Carmen; Linacero, Rosario

    2014-06-18

    The aim of this work was to develop and analytically validate a quantitative RT-PCR method, using novel primer sets designed on Pru du 1, Pru du 3, Pru du 4, and Pru du 6 allergen-coding sequences, and contrast the sensitivity and specificity of these probes. The temperature and/or pressure processing influence on the ability to detect these almond allergen targets was also analyzed. All primers allowed a specific and accurate amplification of these sequences. The specificity was assessed by amplifying DNA from almond, different Prunus species and other common plant food ingredients. The detection limit was 1 ppm in unprocessed almond kernels. The method's robustness and sensitivity were confirmed using spiked samples. Thermal treatment under pressure (autoclave) reduced yield and amplificability of almond DNA; however, high-hydrostatic pressure treatments did not produced such effects. Compared with ELISA assay outcomes, this RT-PCR showed higher sensitivity to detect almond traces in commercial foodstuffs. PMID:24857239

  14. Microtitration plate enzyme immunoassay to detect PCR-amplified DNA from Candida species in blood.

    PubMed Central

    Fujita, S; Lasker, B A; Lott, T J; Reiss, E; Morrison, C J

    1995-01-01

    We developed a microtitration plate enzyme immunoassay to detect PCR-amplified DNA from Candida species. Nucleotide sequences derived from the internal transcribed spacer (ITS) region of fungal rDNA were used to develop species-specific oligonucleotide probes for Candida albicans, C. tropicalis, C. parapsilosis, and C. krusei. No cross-hybridization was detected with any other fungal, bacterial, or human DNAs tested. In contrast, a C. (Torulopsis) glabrata probe cross-reacted with Saccharomyces cerevisiae DNA but with no other DNAs tested. Genomic DNA purified from C. albicans blastoconidia suspended in blood was amplified by PCR with fungus-specific universal primers ITS3 and ITS4. With the C. albicans-specific probe labeled with digoxigenin, a biotinylated capture probe, and streptavidin-coated microtitration plates, amplified DNA from a few as two C. albicans cells per 0.2 ml of blood could be detected by enzyme immunoassay. PMID:7790469

  15. The Genetic Diversity and Evolution of Francisella tularensis with Comments on Detection by PCR.

    PubMed

    Gunnell, Mark K; Adams, Byron J; Robison, Richard A

    2016-01-01

    Francisella tularensis has been the focus of much research over the last two decades mainly because of its potential use as an agent of bioterrorism. F. tularensis is the causative agent of zoonotic tularemia and has a worldwide distribution. The different subspecies of F. tularensis vary in their biogeography and virulence, making early detection and diagnosis important in both the biodefense and public health sectors. Recent genome sequencing efforts reveal aspects of genetic diversity, evolution and phylogeography previously unknown for this relatively small organism, and highlight a role for detection by various PCR assays. This review explores the advances made in understanding the evolution and genetic diversity of F. tularensis and how these advances have led to better PCR assays for detection and identification of the subspecies. PMID:26336102

  16. Aeromonas detection and characterization using genus-specific PCR and single-strand conformation polymorphism (SSCP).

    PubMed

    Delamare, Ana Paula Longaray; Lucena, Roberto Francisco; Thomazi, Guilherme; Ferrarini, Shana; Zacaria, Jucimar; Echeverrigaray, Sergio

    2012-10-01

    Based on sequence alignment, oligonucleotide primers targeting the Aeromonas extracellular lipase gene were developed for PCR detection of member of the genus. A pair of primers designed for conserved regions of the gene amplified a 276 bp sequence in all Aeromonas species and tested strains, but did not have a positive result with other Gram-positive and Gram-negative bacteria, showing high specificity and sensitivity. Selective enrichment in alkaline peptone water, followed by centrifugation, and direct usage of cells suspension as template, detected initial populations of 10 c.f.u. ml⁻¹. Single-strand conformation polymorphism analysis of the PCR products allowed the characterization of Aeromonas strains with a high discriminatory power (Simpson's index = 0.988). The method presented here provides a useful tool for the rapid detection of Aeromonas and the characterization of Aeromonas isolates. PMID:22806741

  17. Rapid detection of virulence factors of Aeromonas isolated from a trout farm by hexaplex-PCR.

    PubMed

    Nam, In-Young; Joh, Kiseong

    2007-08-01

    The detection of virulence factors of Aeromonas is a key component in determining potential pathogenicity because these factors act multifunctionally and multifactorially. In this study water samples were collected from a trout farm on a seasonal basis, and diseased fish and Aeromonas species were isolated and identified. For rapid detection of six virulence factors of isolated Aeromonas, a hexaplex-polymerase chain reaction (hexaplex-PCR) assay was used. The detected virulence factors include aerolysin (aer), GCAT (gcat), serine protease (ser), nuclease (nuc) lipase (lip) and lateral flagella (laf). The dominant strain found in our isolates was Aeromonas sobria, and the dominant virulence factors were aer and nuc for all seasons. We confirmed that A. sobria and two of the virulence genes (aer and nuc) are related. We proposed a method by which one can identify the major strains of Aeromonas: A. hydrophila, A. sobria, A. caviae, and A. veronii, using hexaplex-PCR. PMID:17846582

  18. Molecular detection of Toxoplasma gondii in water samples from Scotland and a comparison between the 529bp real-time PCR and ITS1 nested PCR.

    PubMed

    Wells, Beth; Shaw, Hannah; Innocent, Giles; Guido, Stefano; Hotchkiss, Emily; Parigi, Maria; Opsteegh, Marieke; Green, James; Gillespie, Simon; Innes, Elisabeth A; Katzer, Frank

    2015-12-15

    Waterborne transmission of Toxoplasma gondii is a potential public health risk and there are currently no agreed optimised methods for the recovery, processing and detection of T. gondii oocysts in water samples. In this study modified methods of T. gondii oocyst recovery and DNA extraction were applied to 1427 samples collected from 147 public water supplies throughout Scotland. T. gondii DNA was detected, using real time PCR (qPCR) targeting the 529bp repeat element, in 8.79% of interpretable samples (124 out of 1411 samples). The samples which were positive for T. gondii DNA originated from a third of the sampled water sources. The samples which were positive by qPCR and some of the negative samples were reanalysed using ITS1 nested PCR (nPCR) and results compared. The 529bp qPCR was the more sensitive technique and a full analysis of assay performance, by Bayesian analysis using a Markov Chain Monte Carlo method, was completed which demonstrated the efficacy of this method for the detection of T. gondii in water samples. PMID:26408950

  19. Conventional and PCR Detection of Aphelenchoides fragariae in Diverse Ornamental Host Plant Species

    PubMed Central

    McCuiston, Jamie L.; Hudson, Laura C.; Subbotin, Sergei A.; Davis, Eric L.; Warfield, Colleen Y.

    2007-01-01

    A PCR-based diagnostic assay was developed for early detection and identification of Aphelenchoides fragariae directly in host plant tissues using the species-specific primers AFragFl and AFragRl that amplify a 169-bp fragment in the internal transcribed spacer (ITS1) region of ribosomal DNA. These species-specific primers did not amplify DNA from Aphelenchoides besseyi or Aphelenchoides ritzemabosi. The PCR assay was sensitive, detecting a single nematode in a background of plant tissue extract. The assay accurately detected A. fragariae in more than 100 naturally infected, ornamental plant samples collected in North Carolina nurseries, garden centers and landscapes, including 50 plant species not previously reported as hosts of Aphelenchoides spp. The detection sensitivity of the PCR-based assay was higher for infected yet asymptomatic plants when compared to the traditional, water extraction method for Aphelenchoides spp. detection. The utility of using NaOH extraction for rapid preparation of total DNA from plant samples infected with A. fragariae was demonstrated. PMID:19259510

  20. Quantitative PCR detection of Batrachochytrium dendrobatidis DNA from sediments and water

    USGS Publications Warehouse

    Kirshtein, J.D.; Anderson, C.W.; Wood, J.S.; Longcore, J.E.; Voytek, M.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.

  1. [Real-time PCR Detection Method for the Reston Subtype of the Ebola Virus].

    PubMed

    Xu, Lili; Bao, Linlin; Gu, Songzhi; Qin, Chuan

    2015-05-01

    We aimed to develop a real-time polymerase chain reaction (PCR) detection method for the Reston subtype of the Ebola virus. The NP gene of the Reston subtype of the Ebola virus was selected as the detection object. Sequences of different subtypes of Ebola viruses were aligned using Clustal W software. The most unique and conserved regions of the Reston subtype of the Ebola virus were recruited as candidate sequences for specific primers. Primer Express and Primer Premier 5. 0 software were used to filter the optimal pair of primers for detection. Real-time PCR was carried out using optimized parameters and positive DNA prepared by serial (tenfold) dilution of a recombinant plasmid and by plotting a standard curve. In addition, the reproducibility, accuracy, and specificity of the assay were tested. Results showed that the sensitivity of detection of the Reston subtype of the Ebola virus by real-time PCR could reached 10(2) copies/microL. The linear relationship (R2) reached 0.997, the slope of the standard curve was -0.3101, and amplification efficiency was 110.145%. A sharp and narrow melting peak appeared at 79.94 degrees C for all standards in different dilutions. In conclusion, a fast and sensitive real-time PCR detection system for the Reston subtype of the Ebola virus was developed. This system could be used as a supplementary diagnostic and monitoring approach for basic and clinical studies on the Reston subtype of the Ebola virus. The detection system does not require expensive technology or specialist operators. PMID:26470534

  2. Quantitative PCR for detection of Nosema bombycis in single silkworm eggs and newly hatched larvae.

    PubMed

    Fu, Zhangwuke; He, Xiangkang; Cai, Shunfeng; Liu, Han; He, Xinyi; Li, Mingqian; Lu, Xingmeng

    2016-01-01

    Pebrine disease is the only mandatory quarantine item in sericultural production due to its destructive consequences. So far, the mother moth microscopic examination method established by Pasteur (1870) remains the only detection method for screening for the causative agent Nosema bombycis (N. bombycis). Because pebrine is a horizontal and vertical transmission disease, it is better to inspect silkworm eggs and newly hatched larvae to investigate the infection rate, vertical transmission rate and spore load of the progenies. There is a rising demand for a more direct, effective and accurate detection approach in the sericultural industry. Here, we developed a molecular detection approach based on real-time quantitative PCR (qPCR) for pebrine inspection in single silkworm eggs and newly hatched larvae. Targeting the small-subunit rRNA gene of N. bombycis, this assay showed high sensitivity and reproducibility. Ten spores in a whole sample or 0.1 spore DNA (1 spore DNA represents the DNA content of one N. bombycis spore) in a reaction system was estimated as the detection limit of the isolation and real-time qPCR procedure. Silkworm egg tissues impact the detection sensitivity but are not significant in single silkworm egg detection. Of 400 samples produced by infected moths, 167 and 195 were scored positive by light microscopy and real-time qPCR analysis, respectively. With higher accuracy and the potential capability of high-throughput screening, this method is anticipated to be adaptable for pebrine inspection and surveillance in the sericultural industry. In addition, this method can be applied to ecology studies of N. bombycis-silkworm interactions due to its quantitative function. PMID:26658327

  3. Molecular detection of Mikrocytos mackini in Pacific oysters using quantitative PCR.

    PubMed

    Polinski, Mark; Lowe, Geoff; Meyer, Gary; Corbeil, Serge; Colling, Axel; Caraguel, Charles; Abbott, Cathryn L

    2015-01-01

    Mikrocytos mackini is an internationally regulated pathogen and causative agent of Denman Island disease in Pacific oysters Crassostrea gigas. Recent phylogenetic breakthroughs have placed this parasite within a highly divergent and globally distributed eukaryotic lineage that has been designated a new taxonomic order, Mikrocytida. The discovery of this new radiation of parasites is accompanied by a heightened awareness of the many knowledge gaps that exist with respect to the general biology, epizootiology, and potential impact of mikrocytid parasites on hosts, ecosystems, and commercial fisheries. It has also highlighted current shortcomings regarding our ability to detect these organisms. In this study, we developed a species-specific, sensitive, and quantitative method for detecting M. mackini DNA from host tissues using probe-based real-time qPCR technology. A limit of sensitivity between 2 and 5 genome copy equivalents was achieved in a reaction matrix containing ≥ 40 ng/μL host gDNA without inhibition. This detection proved superior to existing methods based on conventional PCR, histology or gross pathology and is the first species-specific diagnostic test for M. mackini. Quantitative assessment of parasite DNA using this assay remained accurate to between 10 and 50 copies identifying that during infection, M. mackini DNA was significantly more prevalent in hemolymph, labial palp, and mid-body cross-sections compared to mantle or adductor muscle. DNA extracted from a mid-body cross-section also provided the highest likelihood for detection during diagnostic screening of infected oysters. Taken together, these findings provide strong analytical evidence for the adoption of qPCR as the new reference standard for detecting M. mackini and give preliminary insight into the distribution of the parasite within host tissues. Standardised operating methodologies for sample collection and qPCR testing are provided to aid in the international regulatory diagnosis of

  4. Combination of conventional immunohistochemistry and qRT-PCR to detect ALK rearrangement

    PubMed Central

    2014-01-01

    Background Compared with FISH and qRT-PCR analyses, immunohistochemistry (IHC) is the preferred screening test in most pathology practices for ALK-rearrangement detection. With 100% sensitivity and 98% specificity, the VENTANA ALK (D5F3) IHC assay has been approved in the EU and some Asian countries for ALK-rearrangement detection. However, an automated Ventana IHC platform is not available in most pathology labs. In this study, we evaluated the applicability of conventional IHC with D5F3 antibody in routine pathological practice and proposed detection methods and procedures that ensure that patients with ALK+ are not missed. Methods FISH and IHC analyses were performed on 297 lung adenocarcinoma cases. VENTANA IHC and qRT-PCR assay were applied to evaluate ALK-fusion status in the discordant cases of FISH and IHC. The association of ALK+ with clinicopathological characteristics was statistically analyzed. Results IHC had 100% sensitivity and 81.8% specificity for detecting ALK+. Eight ALK-expressed cases were ALK-, five of which had ALK fusion detected by qRT-PCR analysis. Three of these five cases showed ALK expression using VENTANA IHC assay. ALK+ was associated with younger age and lymph node metastasis in this Chinese lung adenocarcinoma patient cohort. Conclusions The advantages of low cost and 100% sensitivity allow conventional IHC to serve as a robust diagnostic tool for screening patients with ALK+, especially in pathology labs without a VENTANA IHC platform. For cases in which ALK is weakly expressed, qRT-PCR is necessary as a diagnostic test for ALK-fusion detection. Virtual slides The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/2269448351088278. PMID:24422905

  5. Fast detection of MYCN copy number alterations in brain neuronal tumors by real-time PCR.

    PubMed

    Malakho, S G; Korshunov, A; Stroganova, A M; Poltaraus, A B

    2008-01-01

    Increased MYCN gene copy number is a characteristic property of neurogenic tumors. Fluorescence in situ hybridization (FISH) and array-based comparative genomic hybridization (array-CGH) are traditionally used to determine MYCN amplification for tumor stratification. A unique ability of real-time quantitative polymerase chain reaction (qPCR) to determine gene copy number, even within a small percent of observed tumor cells, and can be more appropriate. MYCN genomic copy number from 44 human brain tumors (22 medulloblastomas and 22 neurocytomas) was determined by means of FISH, array-CGH, and qPCR. By qPCR, with the original set of oligonucleotides, 17 out of 44 (38.6%) tumors were found to contain a 1.3- to 2.9-fold increase of MYCN defined as low-level gain. An absolute qPCR method was used to get high accuracy of results. Strong correlation was observed between the three methods: for medulloblastomas, r=1 (P<0.01) between FISH and array-CGH and r=0.92 (P<0.01) between qPCR and FISH/array-CGH. For neurocytomas, r=0.9 (P<0.01) between FISH and array-CGH and r=0.34/0.43 (P<0.01) between qPCR and FISH/array-CGH. Absolute qPCR assays possess high precision compared to other conventional methods and can be used for accurate and quickness detection of MYCN status (low-level gene gain and amplification). PMID:18348317

  6. Rapid and Specific Detection of Salmonella spp. in Animal Feed Samples by PCR after Culture Enrichment

    PubMed Central

    Löfström, Charlotta; Knutsson, Rickard; Axelsson, Charlotta Engdahl; Rådström, Peter

    2004-01-01

    A PCR procedure has been developed for routine analysis of viable Salmonella spp. in feed samples. The objective was to develop a simple PCR-compatible enrichment procedure to enable DNA amplification without any sample pretreatment such as DNA extraction or cell lysis. PCR inhibition by 14 different feed samples and natural background flora was circumvented by the use of the DNA polymerase Tth. This DNA polymerase was found to exhibit a high level of resistance to PCR inhibitors present in these feed samples compared to DyNAzyme II, FastStart Taq, Platinum Taq, Pwo, rTth, Taq, and Tfl. The specificity of the Tth assay was confirmed by testing 101 Salmonella and 43 non-Salmonella strains isolated from feed and food samples. A sample preparation method based on culture enrichment in buffered peptone water and DNA amplification with Tth DNA polymerase was developed. The probability of detecting small numbers of salmonellae in feed, in the presence of natural background flora, was accurately determined and found to follow a logistic regression model. From this model, the probability of detecting 1 CFU per 25 g of feed in artificially contaminated soy samples was calculated and found to be 0.81. The PCR protocol was evaluated on 155 naturally contaminated feed samples and compared to an established culture-based method, NMKL-71. Eight percent of the samples were positive by PCR, compared with 3% with the conventional method. The reasons for the differences in sensitivity are discussed. Use of this method in the routine analysis of animal feed samples would improve safety in the food chain. PMID:14711627

  7. Comparative Evaluation of RUT, PCR and ELISA Tests for Detection of Infection with Cytotoxigenic H. pylori

    PubMed Central

    Jalalypour, Farzaneh; Farajnia, Safar; Somi, Mohammad Hossein; Hojabri, Zoya; Yousefzadeh, Rana; Saeedi, Nazli

    2016-01-01

    Purpose: Helicobacter pylori is one of the most prevalent infectious agents in the world which causes a variety of gastrointestinal diseases including gastritis, peptic ulcer and gastric carcinoma. The objective of this study was to comparatively evaluate invasive (rapid urease test and polymerase chain reaction) and non-invasive (enzyme-linked immunosorbent assay) tests in diagnosis of infection with cytotoxigenic H. pylori. Methods: Biopsy specimens and sera were collected from 105 patients with gastric disorders. The presence of H. pylori infection in gastric biopsies was evaluated by RUT and PCR methods using chemotaxis signal transduction protein gene (CSTP), Urea C and HP-16srRNA primers. Serum samples were used for the ELISA test. Detection of infection with cag A-positive strains was performed by PCR and cag A-IgG ELISA kit. Results: Patients with at least two out of three positive results were regarded as infected. The sensitivity, specificity, predictive value and accuracy of the three different methods were evaluated. Of the 105 gastric biopsies, H. pylori were positive in 51 patients (48.57%). The best sensitivity (92.16%) belonged to RUT. The sensitivities of other tests including PCR and ELISA test were 88.24% and 90.20%, respectively. PCR showed the best specificity (94.44%), and the specificities of the other tests including RUT and ELISA test, were 90.74 % and 61.11%, respectively. Furthermore, results of PCR and cag A-IgG ELISA showed high prevalence of cag A-positive strain in the study population. Conclusion: Based on our findings, serum ELISA is a rapid noninvasive test for screening of H. pylori infection in the absence of endoscopy indication. In addition, considering the high prevalence of cytotoxigenic H. pylori strains, cag A is suggested as a promising target for PCR and non- invasive ELISA tests for detection of infection with toxigenic strains. PMID:27478790

  8. Molecular analysis of dolphin morbillivirus: A new sensitive detection method based on nested RT-PCR.

    PubMed

    Centelleghe, Cinzia; Beffagna, Giorgia; Zanetti, Rossella; Zappulli, Valentina; Di Guardo, Giovanni; Mazzariol, Sandro

    2016-09-01

    Cetacean Morbillivirus (CeMV) has been identified as the most pathogenic virus for cetaceans. Over the past three decades, this RNA virus has caused several outbreaks of lethal disease in odontocetes and mysticetes worldwide. Isolation and identification of CeMV RNA is very challenging in whales because of the poor preservation status frequently shown by tissues from stranded animals. Nested reverse transcription polymerase chain reaction (nested RT-PCR) is used instead of conventional RT-PCR when it is necessary to increase the sensitivity and the specificity of the reaction. This study describes a new nested RT-PCR technique useful to amplify small amounts of the cDNA copy of Cetacean morbillivirus (CeMV) when it is present in scant quantity in whales' biological specimens. This technique was used to analyze different tissues (lung, brain, spleen and other lymphoid tissues) from one under human care seal and seven cetaceans stranded along the Italian coastline between October 2011 and September 2015. A well-characterized, 200 base pair (bp) fragment of the dolphin Morbillivirus (DMV) haemagglutinin (H) gene, obtained by nested RT-PCR, was sequenced and used to confirm DMV positivity in all the eight marine mammals under study. In conclusion, this nested RT-PCR protocol can represent a sensitive detection method to identify CeMV-positive, poorly preserved tissue samples. Furthermore, this is also a rather inexpensive molecular technique, relatively easy to apply. PMID:27220282

  9. A Real-Time PCR Method to Detect the Population Level of Halovirus SNJ1.

    PubMed

    Mei, Yunjun; He, Congcong; Deng, Wei; Ba, Dala; Yang, Ming; Zhang, Jian; Zhang, Shunxi; Shen, Ping; Chen, Xiangdong

    2016-01-01

    Although viruses of haloarchaea are the predominant predator in hypersaline ecosystem, the culture studies about halovirus-host systems are infancy. The main reason is the tradition methodology (plaque assay) for virus-host interaction depends on culturable and susceptible host. Actually, more than 90% of haloarchaea are unculturable. Therefore, it is necessary to establish an approach for detecting the dynamics of virus in hypersaline environment without culture. In this study, we report a convenient method to determine the dynamics of halovirus SNJ1 based on quantitative real-time PCR (qPCR). All findings showed that the qPCR method was specific (single peak in melt curves), accurate (a good linear relationship between the log of the PFU and the Ct values, R2 = 0.99), reproducible (low coefficient of variations, below 1%). Additionally, the physicochemical characteristics of the samples tested did not influence the stability of qPCR. Therefore, the qPCR method has the potential value in quantifying and surveying haloviruses in halophilic ecological system. PMID:27192212

  10. Rapid detection of stressed Salmonella spp. in dairy and egg products using immunomagnetic separation and PCR.

    PubMed

    Rijpens, N; Herman, L; Vereecken, F; Jannes, G; De Smedt, J; De Zutter, L

    1999-01-12

    The rapid detection of an average of 5.9 stressed Salmonella cells in 25 g of food product using immunomagnetic separation (IMS) and PCR is described. For pasteurised egg yolk, egg yolk powder, ice-cream, whole egg, egg white and cheeses made from pasteurised milk PCR was applied after 16 h of preenrichment in buffered peptone water (BPW) using IMS and alkaline lysis as sample preparation method. For whole egg and egg white the BPW was supplemented with iron. For milk powder, and raw milk cheeses, the 16-h preenrichment in BPW was followed by IMS and a 4-h enrichment in Rappaport-Vassiliadis broth. In the latter case, PCR was applied on the enrichment medium after centrifugation and alkaline lysis. For PCR the primers ST11 and ST15 (Aabo et al., 1993) producing a fragment of 429 bp were used. An internal PCR control, designed to be co-amplified with the target DNA using the same primers but producing a smaller fragment of 240 bp, was used. PMID:10050683

  11. A Real-Time PCR Method to Detect the Population Level of Halovirus SNJ1

    PubMed Central

    Mei, Yunjun; He, Congcong; Deng, Wei; Ba, Dala; Yang, Ming; Zhang, Jian; Zhang, Shunxi; Shen, Ping; Chen, Xiangdong

    2016-01-01

    Although viruses of haloarchaea are the predominant predator in hypersaline ecosystem, the culture studies about halovirus-host systems are infancy. The main reason is the tradition methodology (plaque assay) for virus-host interaction depends on culturable and susceptible host. Actually, more than 90% of haloarchaea are unculturable. Therefore, it is necessary to establish an approach for detecting the dynamics of virus in hypersaline environment without culture. In this study, we report a convenient method to determine the dynamics of halovirus SNJ1 based on quantitative real-time PCR (qPCR). All findings showed that the qPCR method was specific (single peak in melt curves), accurate (a good linear relationship between the log of the PFU and the Ct values, R2 = 0.99), reproducible (low coefficient of variations, below 1%). Additionally, the physicochemical characteristics of the samples tested did not influence the stability of qPCR. Therefore, the qPCR method has the potential value in quantifying and surveying haloviruses in halophilic ecological system. PMID:27192212

  12. Single-step PCR for detection of Brucella spp. from blood and milk of infected animals.

    PubMed Central

    Leal-Klevezas, D S; Martínez-Vázquez, I O; López-Merino, A; Martínez-Soriano, J P

    1995-01-01

    A versatile method for the extraction of Brucella DNA and PCR are presented as reliable tools for the detection of Brucella spp. from body fluids of infected animals. Two oligonucleotides homologous to regions of the gene encoding for an outer membrane protein (omp-2) were designed to detect the pathogen from milk and/or blood of infected goats, bovines, and human patients. The sensitivity of our test and its ability to detect the pathogen in samples from the field reveal a promising advance in the diagnosis of brucellosis in animals and humans. PMID:8586678

  13. Multiplex nested RT-PCR for detecting avian influenza virus, infectious bronchitis virus and Newcastle disease virus.

    PubMed

    Nguyen, Thanh Trung; Kwon, Hyuk-Joon; Kim, Il-Hwan; Hong, Seung-Min; Seong, Won-Jin; Jang, Jin-Wook; Kim, Jae-Hong

    2013-03-01

    In this study, multiplex nested RT-PCR (mnRT-PCR) was applied to simultaneous detect multiplex PCR with the higher sensitivity of nested PCR that is required for avian influenza, infectious bronchitis and Newcastle disease virus using two steps of amplification. For the first PCR, primers that were specific for each virus were newly designed from the nucleoprotein gene of AIV, the nucleocapsid protein gene of IBV and the fusion protein gene of NDV to amplify products of 665, 386 and 236 nucleotides, respectively. The multiplex PCR step provides mass amplification using common primers, which increased markedly the sensitivity of the test. Non-specific reactions were not observed when other viruses and bacteria were used for evaluating the mnRT-PCR. As a field application, 172 samples were tested by RT-PCR and mnRT-PCR. Among these samples, the concordance rates for mnRT-PCR and the single conventional RT-PCR showed 98.9% (kappa=0.98) and 98.8% (kappa=0.96) similarity for IBV and AIV, respectively. As a result, it is recommended the multiplex nested PCR as an effective tool for detecting and studying the molecular epidemiology of various mixed infections of one or more of these viruses in poultry. PMID:23261801

  14. Single-tube nested PCR for detection of tritrichomonas foetus in feline feces.

    PubMed

    Gookin, Jody L; Birkenheuer, Adam J; Breitschwerdt, Edward B; Levy, Michael G

    2002-11-01

    Tritrichomonas foetus, a venereal pathogen of cattle, was recently identified as an inhabitant of the large intestine in young domestic cats with chronic diarrhea. Recognition of the infection in cats has been mired by unfamiliarity with T. foetus in cats as well as misdiagnosis of the organisms as Pentatrichomonas hominis or Giardia sp. when visualized by light microscopy. The diagnosis of T. foetus presently depends on the demonstration of live organisms by direct microscopic examination of fresh feces or by fecal culturing. As T. foetus organisms are fastidious and fragile, routine flotation techniques and delayed examination and refrigeration of feces are anticipated to preclude the diagnosis in numerous cases. The objective of this study was to develop a sensitive and specific PCR test for the diagnosis of feline T. foetus infection. A single-tube nested PCR was designed and optimized for the detection of T. foetus in feline feces by using a combination of novel (TFITS-F and TFITS-R) and previously described (TFR3 and TFR4) primers. The PCR is based on the amplification of a conserved portion of the T. foetus internal transcribed spacer (ITS) region (ITS1 and ITS2) and the 5.8S rRNA gene. The absolute detection limit of the single-tube nested PCR was 1 organism, while the practical detection limit was 10 organisms per 200 mg of feces. Specificity was examined by using P. hominis, Giardia lamblia, and feline genomic DNA. Our results demonstrate that the single-tube nested PCR is ideally suited for (i) diagnostic testing of feline fecal samples that are found negative by direct microscopy and culturing and (ii) definitive identification of microscopically observable or cultivated organisms. PMID:12409385

  15. Single-Tube Nested PCR for Detection of Tritrichomonas foetus in Feline Feces

    PubMed Central

    Gookin, Jody L.; Birkenheuer, Adam J.; Breitschwerdt, Edward B.; Levy, Michael G.

    2002-01-01

    Tritrichomonas foetus, a venereal pathogen of cattle, was recently identified as an inhabitant of the large intestine in young domestic cats with chronic diarrhea. Recognition of the infection in cats has been mired by unfamiliarity with T. foetus in cats as well as misdiagnosis of the organisms as Pentatrichomonas hominis or Giardia sp. when visualized by light microscopy. The diagnosis of T. foetus presently depends on the demonstration of live organisms by direct microscopic examination of fresh feces or by fecal culturing. As T. foetus organisms are fastidious and fragile, routine flotation techniques and delayed examination and refrigeration of feces are anticipated to preclude the diagnosis in numerous cases. The objective of this study was to develop a sensitive and specific PCR test for the diagnosis of feline T. foetus infection. A single-tube nested PCR was designed and optimized for the detection of T. foetus in feline feces by using a combination of novel (TFITS-F and TFITS-R) and previously described (TFR3 and TFR4) primers. The PCR is based on the amplification of a conserved portion of the T. foetus internal transcribed spacer (ITS) region (ITS1 and ITS2) and the 5.8S rRNA gene. The absolute detection limit of the single-tube nested PCR was 1 organism, while the practical detection limit was 10 organisms per 200 mg of feces. Specificity was examined by using P. hominis, Giardia lamblia, and feline genomic DNA. Our results demonstrate that the single-tube nested PCR is ideally suited for (i) diagnostic testing of feline fecal samples that are found negative by direct microscopy and culturing and (ii) definitive identification of microscopically observable or cultivated organisms. PMID:12409385

  16. Colorimetric Sensor for Label Free Detection of Porcine PCR Product (ID: 18)

    NASA Astrophysics Data System (ADS)

    Ali, M. E.; Hashim, U.; Bari, M. F.; Dhahi, Th. S.

    2011-05-01

    This report described the use of 40±5 nm in diameter citrate-coated gold nanoparticles (GNPs) as colorimetric sensor to visually detect the presence of a 17-base swine specific conserved sequence and nucleotide mismatch in the mixed PCR products of pig, deer and shad cytochrome b genes. The size of these PCR amplicons was 109 base-pair and was amplified with a pair of common primers. Colloidal GNPs changed color from pinkish- red to purple-gray in 2 mM PBS buffer by losing its characteristic surface plasmon resonance peak at 530 nm and gaining new features between 620 and 800 nm in the absorption spectrum indicating strong aggregation. The particles were stabilized against salt induced aggregation, retained spectral features and characteristic color upon adsorption of single-stranded DNA. The PCR products without any additional processing were hybridized with a 17-nucleotide swine probe prior to exposure to GNPs. At a critical annealing temperature (55° C) that differentiated between the match and mismatch pairing, the probe was hybridized with the pig PCR product and dehybridized from the deer's and shad's. The interaction of dehybridized probe to GNPs prevented them from salt-induced aggregation, retaining their characteristic red color. The assay did not need any surface modification chemistry or labeling steps. The results were determined visually and validated by absorption spectroscopy. The entire assay (hybridization plus visual detection) was performed in less than 10 min. The assay obviated the need of complex RFLP, sequencing or blotting to differentiate the same size PCR products. We find the application of the assay for species assignment in food analysis, mismatch detection in genetic screening and homology study among closely related species.

  17. Application of COLD-PCR for improved detection of NF2 mosaic mutations.

    PubMed

    Paganini, Irene; Mancini, Irene; Baroncelli, Marta; Arena, Guido; Gensini, Francesca; Papi, Laura; Sestini, Roberta

    2014-07-01

    Somatic mosaicism represents the coexistence of two or more cell populations with different genotypes in one person, and it is involved in >30 monogenic disorders. Somatic mosaicism characterizes approximately 25% to 33% of patients with de novo neurofibromatosis type 2 (NF2). The identification of mosaicism is crucial to patients and their families because the clinical course of the disease and its transmission risk is influenced by the degree and distribution of mutated cells. Moreover, in NF2, the capability of discriminating patients with mosaicism is especially important to make differential diagnosis with schwannomatosis. However, the identification of mosaic variants is considerably difficult, and the development of specific molecular techniques to detect low levels of unknown molecular alterations is required. Co-amplification at lower denaturation temperature (COLD)-PCR has been described as a powerful method to selectively amplify minority alleles from mixtures of wild-type and mutation-containing sequences. Here, we applied COLD-PCR to molecular analysis of patients with NF2 mosaicism. With the use of COLD-PCR, followed by direct sequencing, we were able to detect NF2 mutations in blood DNA of three patients with NF2 mosaicism. Our study has shown the capability of COLD-PCR in enriching low-represented mutated allele in blood DNA sample, making it usable for molecular diagnosis of patients with mosaicism. PMID:24815379

  18. Rapid detection of MDR-Mycobacterium tuberculosis using modified PCR-SSCP from clinical Specimens

    PubMed Central

    Ali, Imani Fooladi Abbas; Babak, Farzam; Fazlollah, Mousavi Seyed; Nematollah, Jonaidi Jafari

    2014-01-01

    Objective To design a rapid test to detect the rifampin (RIF) and isoniazid (INH) resistant mutant based on polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) technique that analyzes the katG, rpoB genes. Methods Biochemical test as well as IS6110 targeting PCR revealed 103 clinical samples were tuberculosis. To determine the susceptibility of isolates to anti TB drugs, the proportional method was used. Mutations presented within the amplified products of the katG, rpoB genes were evaluated by SSCP. Results Using proportional method, 12 (11.6%) and 9 (8.7%) isolates were resistant respectively to INH and RIF and 9 (8.7%) isolates showed resistance to both drug (multi-drug resistant tuberculosis). Three (2.9%) multi-drug resistant tuberculosis and two INH resistant isolates were detected by the PCR-SSCP and sequencing. The sensitivity and specificity of PCR-SSCP for multi-drug resistant isolates were 33% and 100%, respectively. Conclusions Complete agreement between SSCP and sequencing can indicate that resistance-associated mutations have occurred in other genes except our considered genes. PMID:25183075

  19. Digital gene expression analysis with sample multiplexing and PCR duplicate detection: A straightforward protocol.

    PubMed

    Rozenberg, Andrey; Leese, Florian; Weiss, Linda C; Tollrian, Ralph

    2016-01-01

    Tag-Seq is a high-throughput approach used for discovering SNPs and characterizing gene expression. In comparison to RNA-Seq, Tag-Seq eases data processing and allows detection of rare mRNA species using only one tag per transcript molecule. However, reduced library complexity raises the issue of PCR duplicates, which distort gene expression levels. Here we present a novel Tag-Seq protocol that uses the least biased methods for RNA library preparation combined with a novel approach for joint PCR template and sample labeling. In our protocol, input RNA is fragmented by hydrolysis, and poly(A)-bearing RNAs are selected and directly ligated to mixed DNA-RNA P5 adapters. The P5 adapters contain i5 barcodes composed of sample-specific (moderately) degenerate base regions (mDBRs), which later allow detection of PCR duplicates. The P7 adapter is attached via reverse transcription with individual i7 barcodes added during the amplification step. The resulting libraries can be sequenced on an Illumina sequencer. After sample demultiplexing and PCR duplicate removal with a free software tool we designed, the data are ready for downstream analysis. Our protocol was tested on RNA samples from predator-induced and control Daphnia microcrustaceans. PMID:27401671

  20. Development and validation of a real-time PCR assay for the detection of Aeromonas salmonicida.

    PubMed

    Keeling, S E; Brosnahan, C L; Johnston, C; Wallis, R; Gudkovs, N; McDonald, W L

    2013-05-01

    A real-time PCR assay using a molecular beacon was developed and validated to detect the vapA (surface array protein) gene in the fish pathogen, Aeromonas salmonicida. The assay had 100% analytical specificity and analytical sensitivities of 5 ± 0 fg (DNA), 2.2 × 10(4) ± 1 × 10(4) CFU g(-1) (without enrichment) and 40 ± 10 CFU g(-1) (with enrichment) in kidney tissue. The assay was highly repeatable and proved to be robust following equivalency testing using a different real-time PCR platform. Following analytical validation, diagnostic specificity was determined using New Zealand farmed Chinook salmon, Oncorhynchus tshawytscha (Walbaum), (n = 750) and pink shubunkin, Carassius auratus (L.) (n = 157). The real-time PCR was run in parallel with culture and all fish tested were found to be negative by both methods for A. salmonicida, resulting in 100% diagnostic specificity (95% confidence interval). The molecular beacon real-time PCR system is specific, sensitive and a reproducible method for the detection of A. salmonicida. It can be used for diagnostic testing, health certification and active surveillance programmes. PMID:23121198

  1. A Multiplex PCR-coupled Liquid Bead Array for the Simultaneous Detection of Four Biothreat Agents

    SciTech Connect

    Wilson, W J; Erler, A M; Nasarabadi, S L; Skowronski, E W; McCready, P M

    2004-02-04

    We have developed a 10-plexed PCR assay coupled to a 12-plexed liquid bead array to rapidly screen environmental samples for B. anthracis, Y. pestis, F. tularensis, and B. melitensis. Highly validated species -specific primer sets were used to simultaneously amplify multiple diagnostic regions unique to each individual pathogen. Resolution of the mix of amplified products was achieved by PCR product hybridization to corresponding probe sequences, attached to unique sets of fluorescent beads. The hybridized beads were processed through a flow cytometer, which detected presence and quantity of each PCR product. The assay was optimized to allow for maximum sensitivity in a multiplexed format. A high- throughput demonstration was performed where 384 simulated environmental samples were spiked with different amounts of B. thuringensis spores and pathogen DNA. The samples were robotically processed to extract DNA and arrayed for multiplexed PCR-liquid bead detection. The assay correctly identified the presence or absence of each pathogen and collected over 3,000 individual data points within a single 8-hour shift for approximately $1.20 per sample in a 10-plexed assay.

  2. Quantitative Detection of Clostridium perfringens in the Broiler Fowl Gastrointestinal Tract by Real-Time PCR

    PubMed Central

    Wise, Mark G.; Siragusa, Gregory R.

    2005-01-01

    Strains of Clostridium perfringens are a frequent cause of food-borne disease and gas gangrene and are also associated with necrotic enteritis in chickens. To detect and quantify the levels of C. perfringens in the chicken gastrointestinal tract, a quantitative real-time PCR assay utilizing a fluorogenic, hydrolysis-type probe was developed and utilized to assay material retrieved from the broiler chicken cecum and ileum. Primers and probe were selected following an alignment of 16S rDNA sequences from members of cluster I of the genus Clostridium, and proved to be specific for C. perfringens. The assay could detect approximately 50 fg of C. perfringens genomic DNA and approximately 20 cells in pure culture. Measurements of the analytical sensitivity determined with spiked intestinal contents indicated that the consistent limit of detection with ileal samples was approximately 102 CFU/g of ileal material, but only about 104 CFU/g of cecal samples. The decreased sensitivity with the cecal samples was due to the presence of an unidentified chemical PCR inhibitor(s) in the cecal DNA purifications. The assay was utilized to rapidly detect and quantify C. perfringens levels in the gut tract of broiler chickens reared without supplementary growth-promoting antibiotics that manifested symptoms of necrotic enteritis. The results illustrated that quantitative real-time PCR correlates well with quantification via standard plate counts in samples taken from the ileal region of the gastrointestinal tract. PMID:16000804

  3. Detection of H-1 parvovirus and Kilham rat virus by PCR.

    PubMed Central

    Besselsen, D G; Besch-Williford, C L; Pintel, D J; Franklin, C L; Hook, R R; Riley, L K

    1995-01-01

    H-1 virus and Kilham rat virus (KRV) are autonomous parvoviruses which generally cause subclinical infections in rats and can cause persistent infections in cell cultures. In this study, primer sets specific for either H-1 or KRV were designed on the basis of DNA sequence comparisons of the rodent parvoviruses. The specificities of the H-1 and KRV-specific primer sets were determined by testing viral preparations of seven different parvoviruses and nine other viruses known to infect rodents. The H-1-specific PCR assay amplified the expected 254-bp product only in the presence of H-1 viral DNA and was able to detect as little as 100 fg of H-1 viral DNA. The KRV-specific PCR assay generated the expected 281-bp product only when KRV viral DNA was used as the template and was able to detect as little as 10 pg of KRV viral DNA. Each assay was able to detect its respective virus in tissues from rats experimentally infected with H-1 or KRV. In contrast, no product was amplified by either assay with tissues from mock-infected rats. Our findings indicate that these PCR assays provide rapid, specific, and sensitive methods for the detection of H-1 or KRV infection in rats and cell culture systems. PMID:7665631

  4. Fluorescent detection of Southern blots and PCR-based genetic typing tests

    SciTech Connect

    Mansfield, E.S.; Worley, J.M.; Zimmerman, P.A.

    1994-09-01

    The Southern blot is used to study gene organization, to identify disease-causing genomic rearrangements, or for typing RFLP markers in forensic, paternity, or prenatal diagnostic testing. Fluorescence offers a much greater dynamic range and a more linear response than film used in radioactive or chemiluminescent detection of RFLPs. We therefore investigated using the Fluorimager{trademark} 575 (Molecular Dynamics, Inc.) for analyzing Southern blots. Using a single-locus probe to D2S44 (YNH24) (Promega Corp.), we detect as little as 100 ng (0.05 attomole) genomic DNA. The alkaline phosphatase-labeled probe is detected using AttoPhos (JBL Scientific), and the developed membrane is scanned with the Fluorimager. Biotinylated hybridization probes can also be developed using a streptavidin-alkaline phosphatase conjugate and AttoPhos. The instrument scan parameters can be adjusted to prevent overexposure and accompanying loss of resolution in images of blots, gels, or 96-well microplates. We have used these other sample formats in PCR-based genetic typing assays. We use FluorKit DQS (Molecular Dynamics) to accurately quantify PCR template DNA (1-500 ng) in 96-well microplates scanned using the same instrument. Mutation detection assays run include heteroduplex gels (5% polyacrylamide, 2.7 M urea), short tandem repeat (STR) markers, amplified fragment length polymorphisms (AmpFLP), competitive priming PCR, and allele-specific oligotyping. These assays are run using either 1- or 2-color labeling. We detect unlabeled PCR products, such as the AmpFLP marker D1S80 (Perkin-Elmer) by post-staining gels for 10 minutes with SYBR Green 1 (Molecular Probes) and scanning the wet gel. The Fluorimager scans a 20 x 25 cm sample within three minutes, allowing rapid optimization of fluorescent protocols and high sample throughput.

  5. Association of targeted multiplex PCR with resequencing microarray for the detection of multiple respiratory pathogens.

    PubMed

    Shen, Hongwei; Zhu, Bingqing; Wang, Shulian; Mo, Haolian; Wang, Ji; Li, Jin; Zhang, Chen; Zeng, Huashu; Guan, Li; Shi, Weixian; Zhang, Yong; Ma, Xuejun

    2015-01-01

    A large number of viral and bacterial organisms are responsible for community-acquired pneumonia (CAP) which contributes to substantial burden on health management. A new resequencing microarray (RPM-IVDC1) associated with targeted multiplex PCR was recently developed and validated for multiple respiratory viruses detection and discrimination. In this study, we evaluated the capability of RPM-IVDC1 for simultaneous identification of multiple viral and bacterial organisms. The nasopharyngeal aspirates (NPAs) of 110 consecutive CAP patients, aged from 1 month to 96 years old, were collected from five distinct general hospitals in Beijing during 1-year period. The samples were subjected to the RPM-IVDC1 established protocol as compared to a real-time PCR (qRT-PCR), which was used as standard. The results of virus detection were consistent with those previously described. A total of 37 of Streptococcus pneumoniae, 14 of Haemophilus influenzae, 10 of Mycoplasma pneumoniae, two of Klebsiella pneumoniae and one of Moraxella catarrhalis were detected by RPM-IVDC1. The sensitivities and specificities were compared with those of qRT-PCR for S. pneumoniae (100, 100%, respectively), H. influenzae (92.3, 97.9%, respectively), M. pneumoniae (69.2, 99.0%, respectively), K. pneumoniae (100, 100%, respectively), and M. catarrhalis (100, 100%, respectively). Additional 22 of Streptococcus spp., 24 of Haemophilus spp. and 16 of Neisseria spp. were identified. In addition, methicillin-resistant and carbapenemases allele were also found in nine of Staphylococcus spp. and one of K. pneumoniae, respectively. These results demonstrated the capability of RPM-IVDC1 for simultaneous detection of broad-spectrum respiratory pathogens in complex backgrounds and the advantage of accessing to the actual sequences, showing great potential use of epidemic outbreak investigation. The detection results should be carefully interpreted when introducing this technique in the clinical diagnostics. PMID

  6. Detection of Mycobacterium avium subsp. paratuberculosis by a Direct In Situ PCR Method

    PubMed Central

    Delgado, Fernando; Aguilar, Diana; Garbaccio, Sergio; Francinelli, Gladys; Hernández-Pando, R.; Romano, María Isabel

    2011-01-01

    In situ detection of Mycobacterium avium subsp. paratuberculosis is useful for diagnosis and research of paratuberculosis. The aim of this paper was to detect this agent in formalin-fixed, paraffin-embedded tissue samples by a direct in situ PCR. The technique was performed on ileum or ileocaecal lymph node samples from 8 naturally infected cattle and 1 healthy calf, by using p89 and p92 primers for amplification of IS900 sequence. Moderate positive signal was detected in all positive samples and not in negative control, but tissues resulted were affected in many cases due to the enzymatic treatment and the high temperature exposition. Although the technique was useful for Map detection, the signal was lower than immunohistochemistry probably because of the fixation process. In one case, signal was higher, which might be due to the detection of spheroplasts. Thus, the described method should be recommended when others resulted negative or for spheroplasts detection. PMID:21772965

  7. 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. J. Med. Virol. 88:888-894, 2016. © 2015 Wiley Periodicals, Inc. PMID:26496186

  8. Detection of Cryptosporidium parvum DNA in human feces by nested PCR.

    PubMed Central

    Balatbat, A B; Jordan, G W; Tang, Y J; Silva, J

    1996-01-01

    Cryptosporidium parvum is a coccidian protozoan that causes diarrhea in humans, often chronic and severe in patients with AIDS. Conventionally, diagnosis is made by concentration of stools followed by acid-fast staining (AF) or immunofluorescent staining. The threshold of detection in human stool specimens by these methods may require the presence of 50,000 (immunofluorescent staining) to 500,000 (AF) oocysts per g of stool. In this study, a nested PCR assay was developed to detect C. parvum DNA directly from stool specimens. After extraction of DNA from formalinized stool, a 400-bp fragment of C. parvum DNA was amplified with two 26-mer outer primers. The amplicon from this reaction was amplified with a second primer pair. With these nested primers, a 194-bp DNA fragment was amplified and confirmed as C. parvum DNA by internal probing with an enzyme-linked chemiluminescence system. This PCR-based test allowed the detection of 500 oocysts per g of stool or 100 ng of C. parvum DNA. Studies indicate that the primers utilized are specific for the DNA of C. parvum. DNA sequences were also detected in stool specimens from 4 of 28 patients previously reported negative by AF. In summary, a rapid, sensitive, and specific assay for the detection of C. parvum directly from stool specimens has been developed. This test has the potential for detecting asymptomatic infection, monitoring the response to therapy, and detecting the organism in environmental sources. PMID:8784586

  9. Detection of five potentially periodontal pathogenic bacteria in peri-implant disease: A comparison of PCR and real-time PCR.

    PubMed

    Schmalz, Gerhard; Tsigaras, Sandra; Rinke, Sven; Kottmann, Tanja; Haak, Rainer; Ziebolz, Dirk

    2016-07-01

    The aim of this study was to compare the microbial analysis methods of polymerase chain reaction (PCR) and real-time PCR (RT-PCR) in terms of detection of five selected potentially periodontal pathogenic bacteria in peri-implant disease. Therefore 45 samples of healthy, mucositis and peri-implantitis (n = 15 each) were assessed according to presence of the following bacteria using PCR (DNA-strip technology) and RT-PCR (fluorescent dye SYBR green-system): Aggregatibacter actinomycetemcomitans (Aa), Porphyromonas gingivalis (Pg), Treponema denticola (Td), Tanerella forsythia (Tf), and Fusobacterium nucleatum (Fn). There were no significant correlations between the bacterial and disease patterns, so the benefit of using microbiological tests for the diagnosis of peri-implant diseases is questionable. Correlations between the methods were highest for Tf (Kendall's Tau: 0.65, Spearman: 0.78), Fn (0.49, 0.61) and Td (0.49, 0.59). For Aa (0.38, 0.42) and Pg (0.04, 0.04), lower correlation values were detected. Accordingly, conventional semi-quantitative PCR seems to be sufficient for analyzing potentially periodontal pathogenic bacterial species. PMID:27142589

  10. Evaluation of a novel PCR-based diagnostic assay for detection of Mycobacterium tuberculosis in sputum samples.

    PubMed Central

    Maher, M; Glennon, M; Martinazzo, G; Turchetti, E; Marcolini, S; Smith, T; Dawson, M T

    1996-01-01

    We report on a PCR-based assay we have developed for the detection of Mycobacterium tuberculosis in sputum samples. One hundred sputum specimens, which included 34 culture-positive and 66 culture-negative specimens, were evaluated with this system. Of the 34 culture-positive specimens, 31 were PCR positive, and 60 of the culture-negative specimens were PCR negative. An internal standard has been included in the assay system to monitor PCR inhibition and to confirm the reliability of the PCR assay. PMID:8862607

  11. Validation of qPCR Methods for the Detection of Mycobacterium in New World Animal Reservoirs

    PubMed Central

    Housman, Genevieve; Malukiewicz, Joanna; Boere, Vanner; Grativol, Adriana D.; Pereira, Luiz Cezar M.; Silva, Ita de Oliveira e; Ruiz-Miranda, Carlos R.; Truman, Richard; Stone, Anne C.

    2015-01-01

    Zoonotic pathogens that cause leprosy (Mycobacterium leprae) and tuberculosis (Mycobacterium tuberculosis complex, MTBC) continue to impact modern human populations. Therefore, methods able to survey mycobacterial infection in potential animal hosts are necessary for proper evaluation of human exposure threats. Here we tested for mycobacterial-specific single- and multi-copy loci using qPCR. In a trial study in which armadillos were artificially infected with M. leprae, these techniques were specific and sensitive to pathogen detection, while more traditional ELISAs were only specific. These assays were then employed in a case study to detect M. leprae as well as MTBC in wild marmosets. All marmosets were negative for M. leprae DNA, but 14 were positive for the mycobacterial rpoB gene assay. Targeted capture and sequencing of rpoB and other MTBC genes validated the presence of mycobacterial DNA in these samples and revealed that qPCR is useful for identifying mycobacterial-infected animal hosts. PMID:26571269

  12. Validation of qPCR Methods for the Detection of Mycobacterium in New World Animal Reservoirs.

    PubMed

    Housman, Genevieve; Malukiewicz, Joanna; Boere, Vanner; Grativol, Adriana D; Pereira, Luiz Cezar M; Silva, Ita de Oliveira; Ruiz-Miranda, Carlos R; Truman, Richard; Stone, Anne C

    2015-11-01

    Zoonotic pathogens that cause leprosy (Mycobacterium leprae) and tuberculosis (Mycobacterium tuberculosis complex, MTBC) continue to impact modern human populations. Therefore, methods able to survey mycobacterial infection in potential animal hosts are necessary for proper evaluation of human exposure threats. Here we tested for mycobacterial-specific single- and multi-copy loci using qPCR. In a trial study in which armadillos were artificially infected with M. leprae, these techniques were specific and sensitive to pathogen detection, while more traditional ELISAs were only specific. These assays were then employed in a case study to detect M. leprae as well as MTBC in wild marmosets. All marmosets were negative for M. leprae DNA, but 14 were positive for the mycobacterial rpoB gene assay. Targeted capture and sequencing of rpoB and other MTBC genes validated the presence of mycobacterial DNA in these samples and revealed that qPCR is useful for identifying mycobacterial-infected animal hosts. PMID:26571269

  13. Autonomous Detection of Aerosolized Biological Agents by Multiplexed Immunoassay with PCR Confirmation

    SciTech Connect

    Hindson, B J; McBride, M T; Makarewicz, A J; Henderer, B D; Setlur, U S; Smith, S M; Gutierrez, D M; Metz, T R; Nasarabadi, S L; Venkateswaran, K S; Farrow, S W; Colston, Jr., B W; Dzenitis, J M

    2004-05-27

    The autonomous pathogen detection system (APDS) is an automated, podium-sized instrument that continuously monitors the air for biological threat agents (bacteria, viruses, and toxins). The system has been developed to warn of a biological attack in critical or high-traffic facilities and at special events. The APDS performs continuous aerosol collection, sample preparation, and detection using multiplexed immunoassay followed by confirmatory PCR using real-time TaqMan assays. We have integrated completely reusable flow-through devices that perform DNA extraction and PCR amplification. The fully integrated system was challenged with aerosolized Bacillus anthracis, Yersinia pestis, Bacillus globigii and botulinum toxoid. By coupling highly selective antibody and DNA based assays, the probability of an APDS reporting a false positive is extremely low.

  14. Detection of respiratory syncytial virus by reverse transcription-PCR and hybridization with a DNA enzyme immunoassay.

    PubMed Central

    Freymuth, F; Eugene, G; Vabret, A; Petitjean, J; Gennetay, E; Brouard, J; Duhamel, J F; Guillois, B

    1995-01-01

    Nasal aspirates from 238 infants hospitalized with acute respiratory infections during the winter of 1994 and 1995 were tested for respiratory syncytial virus (RSV) by immunofluorescence assay (IFA) and the viral isolation technique (VIT) and by two PCR and hybridization methods: reverse transcription PCR 1 (RT-PCR1), which amplifies the RNAs of all RSV strains, and RT-PCR-2, which allows subgroup classification of RSV. RT-PCR-1 and RT-PCR-2 detected viral sequences in 56.7% (135 of 238) and 48.3% (115 of 238) of the samples, respectively, while only 80 (33.6%) samples were found to be positive by IFA and VIT. Of the PCR-positive specimens, 57 were missed by these routine techniques in RT-PCR-1 and 45 were missed in RT-PCR-2. Although the RSV-PCR-1 and RSV-PCR-2 techniques amplified two different sequences of the RSV genome, they gave similar results for 218 (91.6%) nasal aspirates. Compared with conventional methods, the sensitivity, specificity, and agreement were 97.5, 63.9, and 75.2%, respectively, for RT-PCR-1 and 89.7, 71.9, and 77.7%, respectively, for RT-PCR-2, and for these two RT-PCR assays, the positive predictive value (PPV) and the index of agreement (kappa) were comparable and moderate, respectively: PPV was 57.8% and kappa was 0.52 in RT-PCR-1, and PPV was 60.9% and kappa was 0.54 in RT-PCR-2. However, there was a perfect correlation between the two RT-PCRs, with a PPV of 100% and an excellent index of agreement (kappa = 0.88). Therefore, most RT-PCR results were really true positive, and VIT and IFA, which missed some of them, appeared to be less sensitive. PMID:8586738

  15. A Quality Control Program within a Clinical Trial Consortium for PCR Protocols To Detect Plasmodium Species

    PubMed Central

    Mayor, Alfredo; Mombo-Ngoma, Ghyslain; Kenguele, Hilaire M.; Ouédraogo, Smaïla; Ndam, Nicaise Tuikue; Mkali, Happy; Mwangoka, Grace; Valecha, Neena; Singh, Jai Prakash Narayan; Clark, Martha A.; Verweij, Jaco J.; Adegnika, Ayola Akim; Severini, Carlo; Menegon, Michela; Macete, Eusebio; Menendez, Clara; Cisteró, Pau; Njie, Fanta; Affara, Muna; Otieno, Kephas; Kariuki, Simon; ter Kuile, Feiko O.; Meshnick, Steven R.

    2014-01-01

    Malaria parasite infections that are only detectable by molecular methods are highly prevalent and represent a potential transmission reservoir. The methods used to detect these infections are not standardized, and their operating characteristics are often unknown. We designed a proficiency panel of Plasmodium spp. in order to compare the accuracy of parasite detection of molecular protocols used by labs in a clinical trial consortium. Ten dried blood spots (DBSs) were assembled that contained P. falciparum, P. vivax, P. malariae, and P. ovale; DBSs contained either a single species or a species mixed with P. falciparum. DBS panels were tested in 9 participating laboratories in a masked fashion. Of 90 tests, 68 (75.6%) were correct; there were 20 false-negative results and 2 false positives. The detection rate was 77.8% (49/63) for P. falciparum, 91.7% (11/12) for P. vivax, 83.3% (10/12) for P. malariae, and 70% (7/10) for P. ovale. Most false-negative P. falciparum results were from samples with an estimated ≤5 parasites per μl of blood. Between labs, accuracy ranged from 100% to 50%. In one lab, the inability to detect species in mixed-species infections prompted a redesign and improvement of the assay. Most PCR-based protocols were able to detect P. falciparum and P. vivax at higher densities, but these assays may not reliably detect parasites in samples with low P. falciparum densities. Accordingly, formal quality assurance for PCR should be employed whenever this method is used for diagnosis or surveillance. Such efforts will be important if PCR is to be widely employed to assist malaria elimination efforts. PMID:24740073

  16. Detection of the oyster herpesvirus in commercial bivalve in northern California, USA: conventional and quantitative PCR.

    PubMed

    Burge, Colleen A; Strenge, Robyn E; Friedman, Carolyn S

    2011-04-01

    The ostreid herpesvirus (OsHV-1) and related oyster herpesviruses (OsHV) are associated with world-wide mortalities of larval and juvenile bivalves. To quantify OsHV viral loads in mollusc tissues, we developed a SYBR Green quantitative PCR (qPCR) based on the A-region of the OsHV-1 genome. Reaction efficiency and precision were demonstrated using a plasmid standard curve. The analytical sensitivity is 1 copy per reaction. We collected Crassostrea gigas, C. sikamea, C. virginica, Ostrea edulis, O. lurida, Mytilus galloprovincialis, and Venerupis phillipinarum from Tomales Bay (TB), and C. gigas from Drakes Estero (DE), California, U.S.A., and initially used conventional PCR (cPCR) to test for presence of OsHV DNA. Subsequently, viral loads were quantified in selected samples of all tested bivalves except O. lurida. Copy numbers were low in each species tested but were significantly greater in C. gigas (p < 0.0001) compared to all other species, suggesting a higher level of infection. OsHV DNA was detected with cPCR and/or qPCR and confirmed by sequencing in C. gigas, C. sikamea, C. virginica, O. edulis, M. galloprovincialis, and V phillipinarum from TB and C. gigas from DE. These data indicate that multiple bivalve species may act as reservoirs for OsHV in TB. A lack of histological abnormalities in potential reservoirs requires alternative methods for their identification. Further investigation is needed to determine the host-parasite relationship for each potential reservoir, including characterization of viral loads and their relationship with infection (via in situ hybridization), assessments of mortality, and host responses. PMID:21648239

  17. Increased efficacy for in-house validation of real-time PCR GMO detection methods.

    PubMed

    Scholtens, I M J; Kok, E J; Hougs, L; Molenaar, B; Thissen, J T N M; van der Voet, H

    2010-03-01

    To improve the efficacy of the in-house validation of GMO detection methods (DNA isolation and real-time PCR, polymerase chain reaction), a study was performed to gain insight in the contribution of the different steps of the GMO detection method to the repeatability and in-house reproducibility. In the present study, 19 methods for (GM) soy, maize canola and potato were validated in-house of which 14 on the basis of an 8-day validation scheme using eight different samples and five on the basis of a more concise validation protocol. In this way, data was obtained with respect to the detection limit, accuracy and precision. Also, decision limits were calculated for declaring non-conformance (>0.9%) with 95% reliability. In order to estimate the contribution of the different steps in the GMO analysis to the total variation variance components were estimated using REML (residual maximum likelihood method). From these components, relative standard deviations for repeatability and reproducibility (RSD(r) and RSD(R)) were calculated. The results showed that not only the PCR reaction but also the factors 'DNA isolation' and 'PCR day' are important factors for the total variance and should therefore be included in the in-house validation. It is proposed to use a statistical model to estimate these factors from a large dataset of initial validations so that for similar GMO methods in the future, only the PCR step needs to be validated. The resulting data are discussed in the light of agreed European criteria for qualified GMO detection methods. PMID:20012027

  18. Evaluation of PCR inhibitory effect of enrichment broths and comparison of DNA extraction methods for detection of Salmonella Enteritidis using real-time PCR assay.

    PubMed

    Hyeon, Ji Yeon; Hwang, In Gyun; Kwak, Hyo Sun; Park, Chankyu; Choi, In Soo; Seo, Kun Ho

    2010-06-01

    The best enrichment broth and DNA extraction scheme was determined for rapid and sensitive detection of Salmonella Enteritidis in steamed pork using real-time PCR. The inhibitory effect of commonly used Salmonella enrichment broths, Rappaport-Vassiliadis (RV) and Muller-Kauffmann tetrathionate with novobiocin (MKTTn), on real-time PCR was confirmed. The inhibition of PCR was statistically significant (p < 0.05) in RV and MKTTn, as compared with buffered peptone water (BPW) or phosphate-buffered saline. The inhibitory effect of the selective enrichment media was successfully removed by using a modified DNA extraction, PrepMan Ultra Reagent with an additional washing step or the DNeasy Tissue Kit. In three experiments, when applied to detection of Salmonella Enteritidis in steamed pork, the real-time PCR coupled with single 24 h enrichment with BPW performed better than double 48 h enrichment with BPW plus RV or MKTTn. The simple real-time PCR assay using BPW proved to be a rapid and sensitive test for detection of low concentrations of Salmonella Enteritidis in steamed pork samples as compared with the conventional culture method. PMID:20458155

  19. A preliminary trial using multi-target polymerase chain reaction (multiplex PCR) and restriction fragment length polymorphism (PCR-RFLP) on the same feedstuffs to detect tissues of animal origin.

    PubMed

    Colombo, F; Marchisio, E; Trezzi, I E; Peri, V; Pinotti, L; Baldi, A; Soncini, G

    2004-08-01

    A preliminary study using multi-target polymerase chain reaction (multiplex PCR) and restriction fragment length polymorphism (PCR-RFLP) was done on the same feedstuffs to detect animal tissues. The results of the two methods differ somewhat: PCR-RFLP did not detect any signal in any sample, but multiplex PCR detected a signal in one sample. These findings could be a basis for further investigations. PMID:15509020

  20. Primer Evaluation for PCR and its Application for Detection of Carbapenemases in Enterobacteriaceae

    PubMed Central

    Mlynarcik, Patrik; Roderova, Magdalena; Kolar, Milan

    2016-01-01

    Background: During the last decade, the prevalence of carbapenem-resistant Enterobacteriaceae in human patients has increased. Carbapenemase-producing bacteria are usually multidrug resistant. Therefore, early recognition of carbapenemase producers is critical to prevent their spread. Objectives: The objective of this study was to develop the primers for single and/or multiplex PCR amplification assays for simultaneous identification of class A, class B, and class D carbapenem hydrolyzing β-lactamases in Enterobacteriaceae and then to evaluate their efficiency. Materials and Methods: The reference sequences of all genes encoding carbapenemases were downloaded from GenBank. Primers were designed to amplify the following 11 genes: blaKPC, blaOXA, blaVIM, blaNDM, blaIMP, blaSME, blaIMI, blaGES, blaGIM, blaDIM and blaCMY. PCR conditions were tested to amplify fragments of different sizes. Two multiplex PCR sets were created for the detection of clinically important carbapenemases. The third set of primers was included for detection of all known carbapenemases in Enterobacteriaceae. They were evaluated using six reference strains and nine clinical isolates. Results: Using optimized conditions, all carbapenemase-positive controls yielded predicted amplicon sizes and confirmed the specificity of the primers in single and multiplex PCR. Conclusions: We have reported here a reliable method, composed of single and multiplex PCR assays, for screening all clinically known carbapenemases. Primers tested in silico and in vitro may distinguish carbapenem-resistant Enterobacteriaceae and could assist in combating the spread of carbapenem resistance in Enterobacteriaceae. PMID:27099689

  1. Real-Time Reverse Transcription-PCR Assay for Comprehensive Detection of Human Rhinoviruses▿

    PubMed Central

    Lu, Xiaoyan; Holloway, Brian; Dare, Ryan K.; Kuypers, Jane; Yagi, Shigeo; Williams, John V.; Hall, Caroline B.; Erdman, Dean D.

    2008-01-01

    Human rhinoviruses (HRVs) are important contributors to respiratory disease, but their healthcare burden remains unclear, primarily because of the lack of sensitive, accurate, and convenient means of determining their causal role. To address this, we developed and clinically validated the sensitivity and specificity of a real-time reverse transcription-PCR (RT-PCR) assay targeting the viral 5′ noncoding region defined by sequences obtained from all 100 currently recognized HRV prototype strains and 85 recently circulating field isolates. The assay successfully amplified all HRVs tested and could reproducibly detect 50 HRV RNA transcript copies, with a dynamic range of over 7 logs. In contrast, a quantified RNA transcript of human enterovirus 68 (HEV68) that showed the greatest sequence homology to the HRV primers and probe set was not detected below a concentration of 5 × 105 copies per reaction. Nucleic acid extracts of 111 coded respiratory specimens that were culture positive for HRV or HEV were tested with the HRV real-time RT-PCR assay and by two independent laboratories that used different in-house HRV/HEV RT-PCR assays. Eighty-seven HRV-culture-positive specimens were correctly identified by the real-time RT-PCR assay, and 4 of the 24 HEV-positive samples were positive for HRV. HRV-specific sequences subsequently were identified in these four specimens, suggesting HRV/HEV coinfection in these patients. The assay was successfully applied in an investigation of a coincidental outbreak of HRV respiratory illness among laboratory staff. PMID:18057136

  2. Failure of PCR to Detect Treponema pallidum ssp. pertenue DNA in Blood in Latent Yaws.

    PubMed

    Marks, Michael; Katz, Samantha; Chi, Kai-Hua; Vahi, Ventis; Sun, Yongcheng; Mabey, David C; Solomon, Anthony W; Chen, Cheng Y; Pillay, Allan

    2015-01-01

    Yaws, caused by Treponema pallidum ssp. pertenue, is a neglected tropical disease closely related to venereal syphilis and is targeted for eradication by 2020. Latent yaws represents a diagnostic challenge, and current tools cannot adequately distinguish between individuals with true latent infection and individuals who are serofast following successful treatment. PCR on blood has previously been shown to detect T. pallidum DNA in patients with syphilis, suggesting that this approach may be of value in yaws. We performed real-time PCR for Treponema pallidum ssp. pertenue on blood samples from 140 children with positive T. pallidum Particle Agglutination (TPPA) and Rapid Plasma Reagin (RPR) tests and 7 controls (negative serology), all collected as part of a prospective study of yaws in the Solomon Islands. All samples were also tested by a nested PCR for T. pallidum. 12 patients had clinical evidence of active yaws whilst 128 were considered to have latent yaws. 43 children had high titre rapid plasma reagins (RPRs) of ≥1:32. PCR testing with both assays gave negative results in all cases. It is possible that the failure to detect T. pallidum ssp. pertenue in blood reflects lower loads of organism in latent yaws compared to those in latent infection with T. pallidum ssp. pertenue, and/or a lower propensity for haematogenous dissemination in yaws than in syphilis. As the goal of the yaws control programme is eradication, a tool that can differentiate true latent infection from individuals who are serofast would be of value; however, PCR of blood is not that tool. PMID:26125585

  3. Failure of PCR to Detect Treponema pallidum ssp. pertenue DNA in Blood in Latent Yaws

    PubMed Central

    Chi, Kai-Hua; Vahi, Ventis; Sun, Yongcheng; Mabey, David C.; Solomon, Anthony W.; Chen, Cheng Y.; Pillay, Allan

    2015-01-01

    Yaws, caused by Treponema pallidum ssp. pertenue, is a neglected tropical disease closely related to venereal syphilis and is targeted for eradication by 2020. Latent yaws represents a diagnostic challenge, and current tools cannot adequately distinguish between individuals with true latent infection and individuals who are serofast following successful treatment. PCR on blood has previously been shown to detect T. pallidum DNA in patients with syphilis, suggesting that this approach may be of value in yaws. We performed real-time PCR for Treponema pallidum ssp. pertenue on blood samples from 140 children with positive T. pallidum Particle Agglutination (TPPA) and Rapid Plasma Reagin (RPR) tests and 7 controls (negative serology), all collected as part of a prospective study of yaws in the Solomon Islands. All samples were also tested by a nested PCR for T. pallidum. 12 patients had clinical evidence of active yaws whilst 128 were considered to have latent yaws. 43 children had high titre rapid plasma reagins (RPRs) of ≥1:32. PCR testing with both assays gave negative results in all cases. It is possible that the failure to detect T. pallidum ssp. pertenue in blood reflects lower loads of organism in latent yaws compared to those in latent infection with T. pallidum ssp. pertenue, and/or a lower propensity for haematogenous dissemination in yaws than in syphilis. As the goal of the yaws control programme is eradication, a tool that can differentiate true latent infection from individuals who are serofast would be of value; however, PCR of blood is not that tool. PMID:26125585

  4. Simultaneous detection and differentiation of Entamoeba histolytica, E. dispar, E. moshkovskii, Giardia lamblia and Cryptosporidium spp. in human fecal samples using multiplex PCR and qPCR-MCA.

    PubMed

    Zebardast, Nozhat; Yeganeh, Farshid; Gharavi, Mohammad Javad; Abadi, Alireza; Seyyed Tabaei, Seyyed Javad; Haghighi, Ali

    2016-10-01

    Entamoeba histolytica, Giardia lamblia and Cryptosporidium spp. are common causes of diarrheal and intestinal diseases all over the world. Microscopic methods are useful in the diagnosis of intestinal parasites (IPs), but their sensitivity was assessed approximately 60 percent. Recently, molecular techniques have been used increasingly for the identification and characterization of the parasites. Among those, in this study we have used multiplex PCR and Real-time PCR with melting curve analysis (qPCR-MCA) for simultaneous detection and differentiation of E. histolytica, E. dispar, E. moshkovskii, G. lamblia and Cryptosporidium spp. in human fecal samples. Twenty DNA samples from 12 E. histolytica and 8 E. dispar samples and twenty stool samples confirmed positive for G. lamblia and Cryptosporidium spp. were analyzed. After DNA extraction from the samples, multiplex PCR was done for detection and differentiation of above mentioned parasites. QPCR-MCA was also performed for the detection and differentiation of 11 isolates of above mentioned parasite in a cycle with a time and temperature. Multiplex PCR was able to simultaneous detect and differentiate of above mentioned parasite in a single reaction. QPCR-MCA was able to differentiate genus and species those five protozoa using melting temperature simultaneously at the same time and temperature programs. In total, qPCR-MCA diagnosed 7/11 isolation of E. histolytica, 6/8 isolation of E. dispar, 1/1 E. moshkovskii Laredo, 10/11 G. Lamblia and 6/11 Cryptosporidium spp. Application of multiplex PCR for detection of more than one species in a test in developing countries, at least in reference laboratories has accurate diagnosis and plays a critical role in differentiation of protozoan species. Multiplex PCR assay with a template and multi template had different results and it seems that using a set of primers with one template has higher diagnostic capability in compare with multi template. The results of this study

  5. Development of SCAR Primers for PCR Assay to Detect Diplodia seriata

    PubMed Central

    Martín, M. T.; Cuesta, M. J.; Martín, L.

    2014-01-01

    The aim of this study was to develop primer pairs for Diplodia seriata identification, one of the most common fungal species associated with grapevine decline in Castilla y León (Spain). Genetic variability of selected isolates of D. seriata was estimated. A molecular marker was generated from a random amplified polymorphic DNA (RAPD) fragment. PCR products of around 1200 bp were obtained with OPE20 primer. The PCR products were cloned and sequenced. The sequences were compared and a fragment of 1207 bp was used to design primer pairs. Two primer pairs were selected (DS3.8 S3-DS3.8 R6 and DS3.8 S3-DS3.8 R4) that amplified a single DNA product of 634 bp and 233 bp, respectively, with D. seriata isolates. No amplification was obtained for any of the 57 isolates of other species. The designed SCAR primer pairs allowed a rapid detection of D. seriata, and were able to detect 0.1 pg of the target DNA. Detection was specific and sensitive for D. seriata. The established protocols detected these fungi in naturally infected grapevines after DNA purification. Diplodia seriata was detectable without DNA purification and isolation in 62.5% to 75% of reactions. The detection of this pathogen in wood samples has great potential for use in pathogen-free certification schemes. PMID:27437468

  6. [Real-time PCR kits for the detection of the African Swine Fever virus].

    PubMed

    Latyshev, O E; Eliseeva, O V; Grebennikova, T V; Verkhovskiĭ, O A; Tsibezov, V V; Chernykh, O Iu; Dzhailidi, G A; Aliper, T I

    2014-01-01

    The results obtained using the diagnostic kit based on real-time polymerase chain reaction to detect the DNA of the African Swine Fever in the pathological material, as well as in the culture fluid, are presented. A high sensitivity and specificity for detection of the DNA in the organs and tissues of animals was shown to be useful for detection in the European Union referentiality reagent kits for DNA detection by real time PCR of ASFV. More rapid and effective method of DNA extraction using columns mini spin Quick gDNA(TM) MiniPrep was suggested and compared to the method of DNA isolation on the inorganic sorbent. High correlation of the results of the DNA detection of ASFV by real-time PCR and antigen detection results ASFV by competitive ELISA obtained with the ELISA SEROTEST/INGEZIM COMRAC PPA was demonstrated. The kit can be used in the veterinary services for effective monitoring of ASFV to contain, eliminate and prevent further spread of the disease. PMID:25895212

  7. Detection and quantification by PCR assay of the biocontrol agent Pantoea agglomerans CPA-2 on apples.

    PubMed

    Soto-Muñoz, Lourdes; Teixidó, Neus; Usall, Josep; Viñas, Inmaculada; Torres, Rosario

    2014-04-01

    The registration of biological control agents requires the development of monitoring systems to detect and quantify the agent in the environment. Pantoea agglomerans CPA-2 is an effective biocontrol agent for postharvest diseases of citrus and pome fruits. The monitoring of CPA-2 in postharvest semi-commercial trials was evaluated by Rodac impression plates and the colonies isolated were confirmed by conventional PCR using the SCAR primers PAGA1 and PAGB1. Samples were taken from different surfaces that had contact with CPA-2, the surrounding environment and working clothes worn by handlers. Moreover, population dynamics of the strain CPA-2 were determined on apple surfaces using both the classical plating technique and real-time quantitative PCR (qPCR). A qPCR assay using a 3'-minor groove-binding (MGB) probe was developed for the specific detection and quantification of P. agglomerans strain CPA-2. Based on the nucleotide sequence of a SCAR fragment of CPA-2, one primer set and TaqMan MGB probe were designed. The primers SP2-F/SP2-R and the TaqMan MGB probe showed a specific detection of strain CPA-2 on apple surfaces, which was verified tested against purified DNA from 17 strains of P. agglomerans, 4 related Pantoea species, and 21 bacterial strains from other genera isolated from whole and also freshly-cut fruit and vegetables. The detection level was approximately 10(3) cells per reaction, and the standard curve was linear within a range of 5log units. Results from semi-commercial trials showed that CPA-2 had a low impact. The maximum persistence of P. agglomerans CPA-2 was not longer than 5days in plastic boxes stored at 0°C. Significant differences in CPA-2 population level dynamics were observed in results obtained by qPCR and dilution plating. These differences may indicate the presence of non-degraded DNA from non-viable cells. In conclusion, qPCR is a novel potential tool to quickly and specifically monitor recent surface colonisation by CPA-2

  8. High-throughput malaria parasite separation using a viscoelastic fluid for ultrasensitive PCR detection.

    PubMed

    Nam, Jeonghun; Shin, Yong; Tan, Justin Kok Soon; Lim, Ying Bena; Lim, Chwee Teck; Kim, Sangho

    2016-05-24

    A novel microfluidic device for high-throughput particle separation using a viscoelastic fluid, which enables the rapid detection of extremely rare malaria parasites by using PCR analysis, is proposed. Our device consists of two segments: the 1st stage for sheathless pre-alignment and the 2nd stage for separation based on size-dependent viscoelasticity-induced lateral migration. The use of a high-aspect ratio channel and a viscoelastic polymer solution with low viscosity enables high-throughput processing. The device performance was first optimized using synthetic particles. A mixture of 2 and 10 μm particles was focused at the center plane in the 1st stage. The smaller particles, serving as surrogates for malaria parasites, were subsequently separated in the 2nd stage with a recovery rate of ∼96% at 400 μl min(-1). Finally, separation of the malaria parasites from the white blood cells was performed. At 400 μl min(-1), almost all white blood cells were removed and the malaria parasites were separated with a ∼94% recovery rate and ∼99% purity. Although the initial concentration of the malaria parasites was too low to be detected by PCR analysis, WBC depletion and buffer removal increased the parasite concentration sufficiently such that PCR detection was possible. PMID:27160315

  9. Difficulties detecting miRNA-203 in human whole saliva by the use of PCR

    PubMed Central

    Lundegard, Martin; Nylander, Karin

    2015-01-01

    Objectives: Oral Lichen Planus (OLP) is a chronic disease of the oral mucosa, and according to the WHO also a pre malignant condition. Micro-RNAs are short non coding RNAs capable of regulating mRNA expression. MiRNA:scan be detected in tissue, blood and human whole saliva (HWS) and recently we have shown miR-203 to be up-regulated in tissue from OLP lesions. Study Design: In order to see whether mRNA as well as miR-203 could be detected also in HWS, saliva from healthy controls and patients with OLP were analysed using two different PCR methods. Results: Results showed low mRNA and miRNA levels in general in HWS samples, making it hard to generate conclusive results. Conclusions: In order to make HWS a valuable source for different analyses, more sensitive PCR techniques capable of detecting very low levels of mRNAand miRNAas well as more efficient methods for extraction of RNA are needed. Key words:miRNA-203, saliva, PCR. PMID:25475777

  10. Real-time immuno-PCR for ultrasensitive detection of pyrene and other homologous PAHs.

    PubMed

    Meng, X Y; Li, Y S; Zhou, Y; Zhang, Y Y; Qiao, B; Sun, Y; Yang, L; Hu, P; Lu, S Y; Ren, H L; Zhang, J H; Wang, X R; Liu, Z S

    2015-08-15

    Polycyclic aromatic hydrocarbons (PAHs) are significant environmental pollutant that can lead to cancer and endocrine system disrupting. Here we developed a real-time immuno-PCR (RT-IPCR) assay based on a biotinylated reporter DNA system for ultrasensitive detection of pyrene (PYR) and homologous PAHs in water. The PAHs in sample compete with PYR-OVA coated on PCR plate to bind with monoclonal antibody (McAb). The biotinylated goat anti-mouse IgG (Bio-IgG) can be captured by the McAb bound with PYR-OVA. Then streptavidin is bound with biotin on Bio-IgG. Finally biotinylated reporter DNA is captured by the streptavidin and quantified by real-time PCR using FastStart universal SYBR Green Master (ROX) kit. The linear range of the assay was from 500 fmol L(-1) to 5 nmol L(-)) with a detection limit of 450 fmol L(-1). The average recoveries of PYR and homologous PAHs from lake water, tap water and commercial mineral water were 96.8%, 101.4% and 99.6% respectively, indicating that water samples had little interfere with the assay. The results demonstrated that the developed RT-IPCR might be a potential method for ultrasensitive detection of PYR and homologous PAHs in drinking and environment water sample. PMID:25791466

  11. A multiplex RT-PCR for the detection of astrovirus, rotavirus, and reovirus in turkeys.

    PubMed

    Jindal, Naresh; Chander, Yogesh; Patnayak, Devi P; Mor, Sunil K; Ziegler, Andre F; Goyal, Sagar M

    2012-09-01

    This study was undertaken to develop and validate a multiplex reverse transcription-polymerase chain reaction (mRT-PCR) for simultaneous detection of avian rotavirus, turkey astrovirus-2 (TAstV-2), and avian reovirus. Primers targeting the conserved regions of NSP4 gene of avian rotavirus, polymerase gene of TAstV-2, and S4 gene of avian reovirus were used. The position of bands at 630, 802, and 1120 base pairs on agarose gel confirmed the presence of rotavirus, TAstV-2, and reovirus, respectively. This mRT-PCR was found to be specific as no amplification was observed with avian influenza virus, Newcastle disease virus, turkey coronavirus, avian metapneumovirus, and intestinal contents of uninfected turkey poults. Intestinal contents of poults from flocks suspected of exhibiting "poult enteritis syndrome" were pooled and tested. Of the 120 pooled samples tested, 70% were positive for TAstV-2, 45% for avian rotavirus, and 18% for avian reovirus. These three viruses were detected alone or in different combinations. Of the samples tested, 20% were negative for these three viruses, 38% were positive for a single virus (TAstV or rotavirus or reovirus), and 42% were positive for two or three viruses. This single-tube mRT-PCR assay has the potential to serve as a rapid diagnostic method for the simultaneous detection of the three enteric viruses in turkeys. PMID:23050480

  12. Detection of canine vector-borne diseases in eastern Poland by ELISA and PCR.

    PubMed

    Dzięgiel, Beata; Adaszek, Łukasz; Carbonero, Alfonso; Łyp, Paweł; Winiarczyk, Mateusz; Dębiak, Piotr; Winiarczyk, Stanisław

    2016-03-01

    The aim of the study was to establish the prevalence of Ehrlichia canis, Anaplasma phagocytophilum and Borrelia burgdorferi in dogs in eastern Poland and to determine the factors associated with exposure (seroposity) or infection (PCR). Anti-A. phagocytophilum, anti-B. burgdorferi and anti-E. canis antibodies were determined in 400 dogs, using the SNAP 4Dx ® test (IDEXX Laboratories). In addition, PCRs were performed for the detection of E. canis, A. phagocytophilum and B. burgdorferi DNA. In reference to the risk factor analysis, a regression logistic model was determined for each aetiological agent. The overall seroprevalence was highest for B. burgdorferi (11.0 %), followed by A. phagocytophilum (8.0 %) and E. canis (1.5 %). Eleven healthy dogs were found to be infected with A. phagocytophilum, as determined by PCR, while the remainder were seronegative. For B. burgdorferi, the DNA of the spirochetes was detected in the blood of 20 dogs, while the presence of anti-B. burgdorferi IgG was detected in the sera of ten of these. For E. canis, none of the dogs tested positive by PCR. Tick control was included as a protective factor for A. phagocytophilum and B. burgdorferi, while the origin (rural) was included as a risk factor for B. burgdorferi and A. phagocytophilum infection. In addition, breed (pure) was a risk factor for B. burgdorferi infection, and sex (female) was a risk factor for E. canis. PMID:26581374

  13. Rapid, multiplex-tandem PCR assay for automated detection and differentiation of toxigenic cyanobacterial blooms.

    PubMed

    Baker, Louise; Sendall, Barbara C; Gasser, Robin B; Menjivar, Toni; Neilan, Brett A; Jex, Aaron R

    2013-01-01

    Cyanobacterial blooms are a major water quality issue and potential public health risk in freshwater, marine and estuarine ecosystems globally, because of their potential to produce cyanotoxins. To date, a significant challenge in the effective management of cyanobacterial has been an inability of classical microscopy-based approaches to consistently and reliably detect and differentiate toxic from non-toxic blooms. The potential of cyanobacteria to produce toxins has been linked to the presence of specific biosynthetic gene clusters. Here, we describe the application of a robotic PCR-based assay for the semi-automated and simultaneous detection of toxin biosynthesis genes of each of the toxin classes characterized to date for cyanobacteria [i.e., microcystins (MCYs), nodularins (NODs), cylindrospermopsins (CYNs) and paralytic shellfish toxins (PSTs)/saxitoxins (SXTs)]. We demonstrated high sensitivity and specificity for each assay using well-characterized, cultured isolates, and establish its utility as a quantitative PCR using DNA, clone and cell-based dilution series. In addition, we used 206 field-collected samples and 100 known negative controls to compare the performance of each assay with conventional PCR and direct toxin detection. We report a diagnostic specificity of 100% and a sensitivity of ≥97.7% for each assay. PMID:23850895

  14. A portable, shock-proof, surface-heated droplet PCR system for Escherichia coli detection.

    PubMed

    Angus, Scott V; Cho, Soohee; Harshman, Dustin K; Song, Jae-Young; Yoon, Jeong-Yeol

    2015-12-15

    A novel polymerase chain reaction (PCR) device was developed that uses wire-guided droplet manipulation (WDM) to guide a droplet over three different heating chambers. After PCR amplification, end-point detection is achieved using a smartphone-based fluorescence microscope. The device was tested for identification of the 16S rRNA gene V3 hypervariable region from Escherichia coli genomic DNA. The lower limit of detection was 10(3) genome copies per sample. The device is portable with smartphone-based end-point detection and provides the assay results quickly (15 min for a 30-cycle amplification) and accurately. The system is also shock and vibration resistant, due to the multiple points of contact between the droplet and the thermocouple and the Teflon film on the heater surfaces. The thermocouple also provides real-time droplet temperature feedback to ensure it reaches the set temperature before moving to the next chamber/step in PCR. The device is equipped to use either silicone oil or coconut oil. Coconut oil provides additional portability and ease of transportation by eliminating spilling because its high melting temperature means it is solid at room temperature. PMID:26164008

  15. Integrated biochip for PCR-based DNA amplification and detection on capacitive biosensors

    NASA Astrophysics Data System (ADS)

    Moschou, D.; Vourdas, N.; Filippidou, M. K.; Tsouti, V.; Kokkoris, G.; Tsekenis, G.; Zergioti, I.; Chatzandroulis, S.; Tserepi, A.

    2013-05-01

    Responding to an increasing demand for LoC devices to perform bioanalytical protocols for disease diagnostics, the development of an integrated LoC device consisting of a μPCR module integrated with resistive microheaters and a biosensor array for disease diagnostics is presented. The LoC is built on a Printed Circuit Board (PCB) platform, implementing both the amplification of DNA samples and DNA detection/identification on-chip. The resistive microheaters for PCR and the wirings for the sensor read-out are fabricated by means of standard PCB technology. The microfluidic network is continuous-flow, designed to perform 30 PCR cycles with heated zones at constant temperatures, and is built onto the PCB utilizing commercial photopatternable polyimide layers. Following DNA amplification, the product is driven in a chamber where a Si-based biosensor array is placed for DNA detection through hybridization. The sensor array is tested for the detection of mutations of the KRAS gene, responsible for colon cancer.

  16. Panfungal PCR and Multiplex Liquid Hybridization for Detection of Fungi in Tissue Specimens

    PubMed Central

    Hendolin, Panu H.; Paulin, Lars; Koukila-Kähkölä, Pirkko; Anttila, Veli-Jukka; Malmberg, Henrik; Richardson, Malcolm; Ylikoski, Jukka

    2000-01-01

    A procedure based on panfungal PCR and multiplex liquid hybridization was developed for the detection of fungi in tissue specimens. The PCR amplified the fungal internal transcribed spacer (ITS) region (ITS1-5.8S rRNA-ITS2). After capture with specific probes, eight common fungal pathogens (Aspergillus flavus, Aspergillus fumigatus, Candida albicans, Candida krusei, Candida glabrata, Candida parapsilosis, Candida tropicalis, and Cryptococcus neoformans) were identified according to the size of the amplification product on an automated sequencer. The nonhybridized products were identified by sequencing. The performance of the procedure was examined with 12 deep-tissue specimens and 8 polypous tissue biopsies from the paranasal sinuses. A detection level of 0.1 to 1 pg of purified DNA (2 to 20 CFU) was achieved. Of the 20 specimens, PCR was positive for 19 (95%), of which 10 (53%) were hybridization positive. In comparison, 12 (60%) of the specimens were positive by direct microscopy, but only 7 (35%) of the specimens showed fungal growth. Sequencing of the nonhybridized amplification products identified an infecting agent in six specimens, and three specimens yielded only sequences of unknown fungal origin. The procedure provides a rapid (within 2 days) detection of common fungal pathogens in tissue specimens, and it is highly versatile for the identification of other fungal pathogens. PMID:11060088

  17. Multiplex Detection of Rare Mutations by Picoliter Droplet Based Digital PCR: Sensitivity and Specificity Considerations

    PubMed Central

    Zonta, Eleonora; Garlan, Fanny; Pécuchet, Nicolas; Perez-Toralla, Karla; Caen, Ouriel; Milbury, Coren; Didelot, Audrey; Fabre, Elizabeth; Blons, Hélène; Laurent-Puig, Pierre; Taly, Valérie

    2016-01-01

    In cancer research, the accuracy of the technology used for biomarkers detection is remarkably important. In this context, digital PCR represents a highly sensitive and reproducible method that could serve as an appropriate tool for tumor mutational status analysis. In particular, droplet-based digital PCR approaches have been developed for detection of tumor-specific mutated alleles within plasmatic circulating DNA. Such an approach calls for the development and validation of a very significant quantity of assays, which can be extremely costly and time consuming. Herein, we evaluated assays for the detection and quantification of various mutations occurring in three genes often misregulated in cancers: the epidermal growth factor receptor (EGFR), the v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) and the Tumoral Protein p53 (TP53) genes. In particular, commercial competitive allele-specific TaqMan® PCR (castPCR™) technology, as well as TaqMan® and ZEN™ assays, have been evaluated for EGFR p.L858R, p.T790M, p.L861Q point mutations and in-frame deletions Del19. Specificity and sensitivity have been determined on cell lines DNA, plasmatic circulating DNA of lung cancer patients or Horizon Diagnostics Reference Standards. To show the multiplexing capabilities of this technology, several multiplex panels for EGFR (several three- and four-plexes) have been developed, offering new "ready-to-use" tests for lung cancer patients. PMID:27416070

  18. A multiplex PCR for the detection of Fasciola hepatica in the intermediate snail host Galba cubensis.

    PubMed

    Alba, Annia; Vázquez, Antonio A; Hernández, Hilda; Sánchez, Jorge; Marcet, Ricardo; Figueredo, Mabel; Sarracent, Jorge; Fraga, Jorge

    2015-07-30

    Fasciolosis is a snail-borne trematode infection that has re-emerged as a human disease, and is considered a significant problem for veterinary medicine worldwide. The evaluation of the transmission risk of fasciolosis as well as the efficacy of the strategies for its control could be carried out through epidemiological surveillance of the snails that act as intermediate hosts of the parasites. The present study aimed to develop the first multiplex PCR to detect Fasciola hepatica in Galba cubensis, an important intermediate host of the parasite in the Americas and especially in the Caribbean basin. The multiplex PCR was optimized for the amplification of a 340 bp fragment of the second internal transcribed spacer (ITS-2) of F. hepatica rDNA, while another set of primers was designed and used to amplify a conserved segment of the nuclear 18S rDNA of the snail (451 bp), as an internal control of the reaction. The assay was able to detect up to 100 pg of the parasite even at high concentrations of snail DNA, an analytical sensitivity that allows the detection of less than a single miracidium, which is the minimal biological infestation unit. A controlled laboratory-reared G. cubensis - F. hepatica system was used for the evaluation of the developed multiplex PCR, and 100% sensitivity and specificity was achieved. This assay constitutes a novel, useful and suitable technique for the survey of fasciolosis transmission through one of the main intermediate hosts in the Western hemisphere. PMID:26012858

  19. Rapid detection of Actinobacillus actinomycetemcomitans, Prevotella intermedia and Porphyromona gingivalis by multiplex PCR.

    PubMed

    García, L; Tercero, J C; Legido, B; Ramos, J A; Alemany, J; Sanz, M

    1998-01-01

    The identification of specific periodontal pathogens by conventional methods, mainly anaerobic cultivation, is difficult, time consuming and even sometimes unreliable. Therefore, a multiplex PCR method for simultaneous detection of Actinobacillus actinomycetemcomitans (A.a.), Porphyromona gingivalis (P.g.) and Prevotella intermedia (P.i.) was developed for rapid and easy identification of these specific bacterial pathogens in subgingival plaque samples. In this paper, there is a detailed description of the oligonucleotide primer selection, DNA extraction and PCR conditions and the sequencing of the amplified products. The locus chosen to be amplified is a highly variable region in the 16S ribosomal DNA. For the development of this technique ATCC cultures and pure cultures from subgingival plaque samples taken from periodontitis patients were used. As an internal positive control a recombinant plasmid was developed. This simple DNA extraction procedure and the DNA amplification and visualization of the amplified product permits the detection of the bacteria in a working day. Thus, this multiplex PCR method is a rapid and effective detection method for specific periodontal pathogens. PMID:9524322

  20. Detection of Rare Drug Resistance Mutations by Digital PCR in a Human Influenza A Virus Model System and Clinical Samples

    PubMed Central

    Bushell, Claire A.; Grant, Paul R.; Cowen, Simon; Gutierrez-Aguirre, Ion; O'Sullivan, Denise M.; Žel, Jana; Milavec, Mojca; Foy, Carole A.; Nastouli, Eleni; Garson, Jeremy A.; Huggett, Jim F.

    2015-01-01

    Digital PCR (dPCR) is being increasingly used for the quantification of sequence variations, including single nucleotide polymorphisms (SNPs), due to its high accuracy and precision in comparison with techniques such as quantitative PCR (qPCR) and melt curve analysis. To develop and evaluate dPCR for SNP detection using DNA, RNA, and clinical samples, an influenza virus model of resistance to oseltamivir (Tamiflu) was used. First, this study was able to recognize and reduce off-target amplification in dPCR quantification, thereby enabling technical sensitivities down to 0.1% SNP abundance at a range of template concentrations, a 50-fold improvement on the qPCR assay used routinely in the clinic. Second, a method was developed for determining the false-positive rate (background) signal. Finally, comparison of dPCR with qPCR results on clinical samples demonstrated the potential impact dPCR could have on clinical research and patient management by earlier (trace) detection of rare drug-resistant sequence variants. Ultimately this could reduce the quantity of ineffective drugs taken and facilitate early switching to alternative medication when available. In the short term such methods could advance our understanding of microbial dynamics and therapeutic responses in a range of infectious diseases such as HIV, viral hepatitis, and tuberculosis. Furthermore, the findings presented here are directly relevant to other diagnostic areas, such as the detection of rare SNPs in malignancy, monitoring of graft rejection, and fetal screening. PMID:26659206

  1. Detection of Rare Drug Resistance Mutations by Digital PCR in a Human Influenza A Virus Model System and Clinical Samples.

    PubMed

    Whale, Alexandra S; Bushell, Claire A; Grant, Paul R; Cowen, Simon; Gutierrez-Aguirre, Ion; O'Sullivan, Denise M; Žel, Jana; Milavec, Mojca; Foy, Carole A; Nastouli, Eleni; Garson, Jeremy A; Huggett, Jim F

    2016-02-01

    Digital PCR (dPCR) is being increasingly used for the quantification of sequence variations, including single nucleotide polymorphisms (SNPs), due to its high accuracy and precision in comparison with techniques such as quantitative PCR (qPCR) and melt curve analysis. To develop and evaluate dPCR for SNP detection using DNA, RNA, and clinical samples, an influenza virus model of resistance to oseltamivir (Tamiflu) was used. First, this study was able to recognize and reduce off-target amplification in dPCR quantification, thereby enabling technical sensitivities down to 0.1% SNP abundance at a range of template concentrations, a 50-fold improvement on the qPCR assay used routinely in the clinic. Second, a method was developed for determining the false-positive rate (background) signal. Finally, comparison of dPCR with qPCR results on clinical samples demonstrated the potential impact dPCR could have on clinical research and patient management by earlier (trace) detection of rare drug-resistant sequence variants. Ultimately this could reduce the quantity of ineffective drugs taken and facilitate early switching to alternative medication when available. In the short term such methods could advance our understanding of microbial dynamics and therapeutic responses in a range of infectious diseases such as HIV, viral hepatitis, and tuberculosis. Furthermore, the findings presented here are directly relevant to other diagnostic areas, such as the detection of rare SNPs in malignancy, monitoring of graft rejection, and fetal screening. PMID:26659206

  2. A multiplex real-time PCR panel assay for simultaneous detection and differentiation of 12 common swine viruses.

    PubMed

    Shi, Xiju; Liu, Xuming; Wang, Qin; Das, Amaresh; Ma, Guiping; Xu, Lu; Sun, Qing; Peddireddi, Lalitha; Jia, Wei; Liu, Yanhua; Anderson, Gary; Bai, Jianfa; Shi, Jishu

    2016-10-01

    Mixed infection with different pathogens is common in swine production systems especially under intensive production conditions. Quick and accurate detection and differentiation of different pathogens are necessary for epidemiological surveillance, disease management and import and export controls. In this study, we developed and validated a panel of multiplex real-time PCR/RT-PCR assays composed of four subpanels, each detects three common swine pathogens. The panel detects 12 viruses or viral serotypes, namely, VSV-IN, VSV-NJ, SVDV, CSFV, ASFV, FMDV, PCV2, PPV, PRV, PRRSV-NA, PRRSV-EU and SIV. Correlation coefficients (R(2)) and PCR amplification efficiencies of all singular and triplex real-time PCR reactions are within the acceptable range. Comparison between singular and triplex real-time PCR assays of each subpanel indicates that there is no significant interference on assay sensitivities caused by multiplexing. Specificity tests on 226 target clinical samples or 4 viral strains and 91 non-target clinical samples revealed that the real-time PCR panel is 100% specific, and there is no cross amplification observed. The limit of detection of each triplex real-time PCR is less than 10 copies per reaction for DNA, and less than 16 copies per reaction for RNA viruses. The newly developed multiplex real-time PCR panel also detected different combinations of co-infections as confirmed by other means of detections. PMID:27506582

  3. PrimerMapper: high throughput primer design and graphical assembly for PCR and SNP detection.

    PubMed

    O'Halloran, Damien M

    2016-01-01

    Primer design represents a widely employed gambit in diverse molecular applications including PCR, sequencing, and probe hybridization. Variations of PCR, including primer walking, allele-specific PCR, and nested PCR provide specialized validation and detection protocols for molecular analyses that often require screening large numbers of DNA fragments. In these cases, automated sequence retrieval and processing become important features, and furthermore, a graphic that provides the user with a visual guide to the distribution of designed primers across targets is most helpful in quickly ascertaining primer coverage. To this end, I describe here, PrimerMapper, which provides a comprehensive graphical user interface that designs robust primers from any number of inputted sequences while providing the user with both, graphical maps of primer distribution for each inputted sequence, and also a global assembled map of all inputted sequences with designed primers. PrimerMapper also enables the visualization of graphical maps within a browser and allows the user to draw new primers directly onto the webpage. Other features of PrimerMapper include allele-specific design features for SNP genotyping, a remote BLAST window to NCBI databases, and remote sequence retrieval from GenBank and dbSNP. PrimerMapper is hosted at GitHub and freely available without restriction. PMID:26853558

  4. PrimerMapper: high throughput primer design and graphical assembly for PCR and SNP detection

    PubMed Central

    O’Halloran, Damien M.

    2016-01-01

    Primer design represents a widely employed gambit in diverse molecular applications including PCR, sequencing, and probe hybridization. Variations of PCR, including primer walking, allele-specific PCR, and nested PCR provide specialized validation and detection protocols for molecular analyses that often require screening large numbers of DNA fragments. In these cases, automated sequence retrieval and processing become important features, and furthermore, a graphic that provides the user with a visual guide to the distribution of designed primers across targets is most helpful in quickly ascertaining primer coverage. To this end, I describe here, PrimerMapper, which provides a comprehensive graphical user interface that designs robust primers from any number of inputted sequences while providing the user with both, graphical maps of primer distribution for each inputted sequence, and also a global assembled map of all inputted sequences with designed primers. PrimerMapper also enables the visualization of graphical maps within a browser and allows the user to draw new primers directly onto the webpage. Other features of PrimerMapper include allele-specific design features for SNP genotyping, a remote BLAST window to NCBI databases, and remote sequence retrieval from GenBank and dbSNP. PrimerMapper is hosted at GitHub and freely available without restriction. PMID:26853558

  5. Two RT-PCR based assays to detect human metapneumovirus in nasopharyngeal aspirates.

    PubMed

    López-Huertas, María Rosa; Casas, Inmaculada; Acosta-Herrera, Belsy; García, María Luz; Coiras, María Teresa; Pérez-Breña, Pilar

    2005-10-01

    Two sensitive and specific RT-PCR assays were standardised for testing the presence of human metapneumovirus. A total of 300 nasopharyngeal aspirates collected from infants suffering from bronchiolitis since October 2000 to June 2003 and shown previously as negative to common respiratory viruses were examined. Matrix and polymerase viral genes, which show a low rate of variation, were chosen to design amplification assays to ensure that any genotype of the human metapneumovirus could be detected. A RT-PCR followed by a reverse line blotting hybridisation was developed for viral polymerase gene. For the matrix gene, after the RT-PCR assay, a subsequent nested PCR was carried out. Both assays had similar sensitivity, equivalent to 0.1 TCID50 of human metapneumovirus strain NL/1/99 which was used as the positive control. The human metapneumovirus was present in 16.6% of the specimens studied. The approaches described below are not only a robust method for rapid diagnosis of the human metapneumovirus, but also to establish an etiological surveillance tool for epidemiological studies. Based on the results obtained, human metapneumovirus infections in Madrid followed a seasonal pattern, with most of the infections occurring between February and April. PMID:15961167

  6. Diagnostic Value of PCR for Detection of Borrelia burgdorferi in Skin Biopsy and Urine Samples from Patients with Skin Borreliosis

    PubMed Central

    Brettschneider, S.; Bruckbauer, H.; Klugbauer, N.; Hofmann, H.

    1998-01-01

    Skin biopsies of 36 patients with erythema migrans and acrodermatitis chronica atrophicans (ACA) before therapy and those of 8 patients after therapy were examined for Borrelia burgdorferi DNA by PCR. Skin biopsies of 27 patients with dermatological diseases other than Lyme borreliosis and those of 10 healthy persons were examined as controls. Two different primer sets targeting 23S rRNA (PCR I) and 66-kDa protein (PCR II) genes were used. PCR was performed with freshly frozen tissue (FFT) and paraffin-embedded tissue (PET). For FFT specimens of erythema migrans, 73% were positive by PCR I, 79% were positive by PCR II, and 88% were positive by combining PCR I and II. For PET specimens, PCR was less sensitive (PCR I, 44%; PCR II, 52%). For FFT specimens of ACA, PCR I was positive for two of five patients and PCR II was positive for four of five patients. B. burgdorferi was cultured from 79% of the erythema migrans specimens but not from any of the ACA lesions. Elevated B. burgdorferi antibodies were detected in sera of 74% of erythema migrans patients and 100% of ACA patients. All urine samples were negative by PCR II, whereas PCR I was positive for 27%. However, hybridization of these amplicons was negative. Sequencing of three amplicons identified nonborrelial DNA. In conclusion, urine PCR is not suitable for the diagnosis of skin borreliosis. A combination of two different primer sets achieves high sensitivity with skin biopsies. In early erythema migrans infection, culture and PCR are more sensitive than serology. PMID:9705410

  7. Rapid detection of Listeria monocytogenes by real-time PCR in processed meat and dairy products.

    PubMed

    Heo, Eun Jeong; Song, Bo Ra; Park, Hyun Jung; Kim, Young Jo; Moon, Jin San; Wee, Sung Hwan; Kim, Jin-Seok; Yoon, Yohan

    2014-03-01

    The objectives of this study were to evaluate the detection of Listeria monocytogenes in different ready-to-eat foods using real-time PCR (RT-PCR). Various concentrations (10(0) to 10(5) CFU/ml) of L. monocytogenes ATCC 19115 were inoculated into ham, sausage, ground meat, processed milk, cheese, and infant formula. L. monocytogenes ATCC 19115 in the samples was then enumerated on Oxford agar, and DNA was extracted from the samples before and after incubation at 36°C for 4 h. A set of primers and hybridization probe designed in this study was then used to detect the pathogen. The standard curve was then prepared by plotting cycle threshold values for each dilution versus L. monocytogenes cell counts (log CFU). The specificity of the set of primers and hybridization probe was appropriate. A 4-h incubation at 36°C before DNA extraction produced optimum standard curves in comparison to the results for a 0-h incubation. Thus, a 4-h incubation at 36°C was applied for monitoring L. monocytogenes in collected food samples. To monitor L. monocytogenes in foods, 533 samples (ham, 129; sausage, 226; ground meat, 72; processed cheese, 54; processed milk, 42; and infant formula, 10) were collected from retail markets and from the step before pasteurization in plants. Of all 533 samples, 4 samples (0.8%) showed positive signals in RT-PCR. Two samples from hams (1.6%) and two samples from sausages (0.9%) were determined to be positive for L. monocytogenes at < 100 CFU/g. The results indicate that the RT-PCR detection method with the set of primers and hybridization probe designed in this study should be useful in monitoring for L. monocytogenes in processed meat and milk products. PMID:24674437

  8. Quick detection of Leifsonia xyli subsp. xyli by PCR and necleotide sequence analysis of PCR amplicons from Chinese Leifsonia xyli subsp. xyli isolates

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A quick polymerase chain reaction (PCR) assay was developed for the detection of Leifsonia xyli subsp. xyli (Lxx), the bacterial causal agent of ratoon stunting disease (RSD) of sugarcane, in crude juice samples from stalks. After removal of abiotic impurities and large molecular weight microorgani...

  9. Detection of Banana mild mosaic virus and Banana virus X by polyvalent degenerate oligonucleotide RT-PCR (PDO-RT-PCR).

    PubMed

    Teycheney, Pierre-Yves; Acina, Isabelle; Lockhart, Benham E L; Candresse, Thierry

    2007-06-01

    Viruses are important constraints to the movement and propagation of plant germplasm, especially for vegetatively propagated crops such as banana and plantain. Their control relies primarily on the use of virus-free plant material, whose production and certification requires sensitive and reliable detection methods. An existing polyvalent degenerate oligonucleotide RT-PCR (PDO-RT-PCR) assay was adapted to the detection of Banana mild mosaic virus (BanMMV) and Banana virus X, two Flexiviridae infecting Musa spp. PDO inosine-containing primers were found to be well suited to the detection of BanMMV, despite its high molecular diversity, but not to that of the highly conserved BVX, for which species-specific primers were designed. Sampling and sample processing steps were optimized in order to avoid nucleic acid purification prior to the reverse transcription step. A polyclonal anti-BanMMV antiserum was raised and successfully used for the immunocapture (IC) of BanMMV viral particles from leaf extracts, leading to the development of a PDO-IC-RT-nested PCR assay. Although the anti-BanMMV antiserum could to some extent recognize BVX viral particles, direct binding (DB) was shown to be a more efficient method for processing BVX-infected samples and a PDO-DB-RT-nested PCR assay was developed for the detection of BVX from leaf extracts. PMID:17280722

  10. Evaluation of a Modular Multiplex-PCR Methicillin-Resistant Staphylococcus aureus Detection Assay Adapted for mecC Detection

    PubMed Central

    Larsen, Anders R.; Skov, Robert L.; Paterson, Gavin K.; Holmes, Mark A.; Sabat, Artur J.; Friedrich, Alexander W.; Köck, Robin; Peters, Georg; Kriegeskorte, André

    2013-01-01

    A mecC (mecALGA251)-adapted multiplex PCR-based methicillin-resistant Staphylococcus aureus (MRSA) detection assay was evaluated using an international, spa-typed Staphylococcus aureus collection comprising 51 mecC-positive MRSA, 240 mecA-positive MRSA, and 50 mecA- and mecC-negative methicillin-susceptible S. aureus (MSSA) isolates. The assay showed 100% sensitivity and specificity for S. aureus species identification as well as for mecA and mecC detection. PMID:23515551

  11. SYBR Green Real-Time PCR Method To Detect Clostridium botulinum Type A▿

    PubMed Central

    Fenicia, Lucia; Anniballi, Fabrizio; De Medici, Dario; Delibato, Elisabetta; Aureli, Paolo

    2007-01-01

    Botulinum toxins (BoNTs) are classically produced by Clostridium botulinum but rarely also from neurotoxigenic strains of Clostridium baratii and Clostridium butyricum. BoNT type A (BoNT/A), BoNT/B, BoNT/E, and very rarely BoNT/F are mainly responsible for human botulism. Standard microbiological methods take into consideration only the detection of C. botulinum. The presumptive identification of the toxigenic strains together with the typing of BoNT has to be performed by mouse bioassay. The development of PCR-based methods for the detection and typing of BoNT-producing clostridia would be an ideal alternative to the mouse bioassay. The objective of this study was to develop a rapid and robust real-time PCR method for detecting C. botulinum type A. Four different techniques for the extraction and purification of DNA from cultured samples were initially compared. Of the techniques used, Chelex 100, DNeasy tissue kit, InstaGene matrix DNA, and boiling, the boiling technique was significantly less efficient than the other three. These did not give statistically different results, and Chelex 100 was chosen because it was less expensive than the others. In order to eliminate any false-negative results, an internal amplification control was synthesized and included in the amplification mixture according to ISO 22174. The specificity of the method was tested against 75 strains of C. botulinum type A, 4 strains of C. botulinum type Ab, and 101 nontarget strains. The detection limit of the reaction was less than 6 × 101 copies of C. botulinum type A DNA. The robustness of the method was confirmed using naturally contaminated stool specimens to evaluate the tolerance of inhibitor substances. SYBR green real-time PCR showed very high specificity for the detection of C. botulinum types A and Ab (inclusivity and exclusivity, 100%). PMID:17369349

  12. SYBR green real-time PCR method to detect Clostridium botulinum type A.

    PubMed

    Fenicia, Lucia; Anniballi, Fabrizio; De Medici, Dario; Delibato, Elisabetta; Aureli, Paolo

    2007-05-01

    Botulinum toxins (BoNTs) are classically produced by Clostridium botulinum but rarely also from neurotoxigenic strains of Clostridium baratii and Clostridium butyricum. BoNT type A (BoNT/A), BoNT/B, BoNT/E, and very rarely BoNT/F are mainly responsible for human botulism. Standard microbiological methods take into consideration only the detection of C. botulinum. The presumptive identification of the toxigenic strains together with the typing of BoNT has to be performed by mouse bioassay. The development of PCR-based methods for the detection and typing of BoNT-producing clostridia would be an ideal alternative to the mouse bioassay. The objective of this study was to develop a rapid and robust real-time PCR method for detecting C. botulinum type A. Four different techniques for the extraction and purification of DNA from cultured samples were initially compared. Of the techniques used, Chelex 100, DNeasy tissue kit, InstaGene matrix DNA, and boiling, the boiling technique was significantly less efficient than the other three. These did not give statistically different results, and Chelex 100 was chosen because it was less expensive than the others. In order to eliminate any false-negative results, an internal amplification control was synthesized and included in the amplification mixture according to ISO 22174. The specificity of the method was tested against 75 strains of C. botulinum type A, 4 strains of C. botulinum type Ab, and 101 nontarget strains. The detection limit of the reaction was less than 6 x 10(1) copies of C. botulinum type A DNA. The robustness of the method was confirmed using naturally contaminated stool specimens to evaluate the tolerance of inhibitor substances. SYBR green real-time PCR showed very high specificity for the detection of C. botulinum types A and Ab (inclusivity and exclusivity, 100%). PMID:17369349

  13. Unreliability of results of PCR detection of Helicobacter pylori in clinical or environmental samples.

    PubMed

    Sugimoto, Mitsushige; Wu, Jeng-Yih; Abudayyeh, Suhaib; Hoffman, Jill; Brahem, Hajer; Al-Khatib, Khaldun; Yamaoka, Yoshio; Graham, David Y

    2009-03-01

    The aim of this study was to compare published Helicobacter pylori primer pairs for their ability to reliably detect H. pylori in gastric biopsy specimens and salivary samples. Detection limits of the 26 PCR primer pairs previously described for detection of H. pylori DNA in clinical samples were determined. Sensitivity and specificity were determined using primers with detection limits of <100 CFU/ml using 50 H. pylori-positive and -negative (by concordance by culture and histology) coded gastric biopsy specimens. These results were then confirmed with gastric biopsy specimens and saliva from patients with confirmed H. pylori status. Five of the twenty-six previously reported primer pairs (HP64-f/HP64-r, HP1/HP2, EHC-U/EHC-L, VAG-F/VAG-R, and ICT37/ICT38) had detection limits of <100 CFU/ml in the presence of gastric tissue. None had 100% specificity or sensitivity; all produced false-positive results. The HP64-f/HP64-r for ureA and HP1/HP2 for 16S rRNA individually had sensitivities and specificities of >90% with gastric biopsy specimens. No combinations of primer pairs improved the results. Using these five primer pairs, 54% of the positive saliva samples were determined to be false positive; both the HP64-f/HP64-r and the HP1/HP2 sets produced false positives with saliva. We conclude that clinicians should not rely on results using current PCR primers alone to decide the H. pylori status of an individual patient or as a basis for treatment decisions. The results of studies based on PCR identification of H. pylori in environmental samples should be viewed with caution. Possibly, specific primers sets can be identified based on the presence of multiple putative virulence factor genes. PMID:19129407

  14. A TaqMan-based real-time PCR for detection and quantification of porcine parvovirus 4.

    PubMed

    Gava, Danielle; Souza, Carine K; Schaefer, Rejane; Vincent, Amy L; Cantão, Maurício E; Coldebella, Arlei; Ciacci-Zanella, Janice R

    2015-07-01

    Porcine parvovirus 4 (PPV4) is a DNA virus, and a member of the Parvoviridae family within the Bocavirus genera. It was detected recently in swine, but its epidemiology and pathology remain unclear. A TaqMan-based real-time PCR (qPCR) targeting a conserved region of the ORF3 gene of PPV4 was developed. The qPCR detection limit was 9.5 × 10(1) DNA copies/μL. There was no cross-reaction with porcine parvovirus, torque teno virus 1, torque teno virus 2, porcine circovirus type 1, porcine circovirus type 2, or with pseudorabies virus. Two hundred and seventy-two samples, including serum, semen, lungs, feces, ovarian follicular fluids, ovaries and uterus, were evaluated by qPCR and PPV4 was detected in 36 samples (13.2%). When compared with a conventional PCR (cPCR), the qPCR assay was 10 times more sensitive and the detection of PPV4 DNA in field samples was increased 2.5 times. Partial sequencing of PPV4 ORF3 gene, obtained from two pooled samples of uterus and ovaries, revealed a high nucleotide identity (98-99%) with a reference PPV4 sequence. The qPCR can be used as a fast and accurate assay for the detection and quantification of PPV4 in field samples and for epidemiological studies in swine herds. PMID:25813599

  15. A multiplex nested PCR for the detection and identification of Candida species in blood samples of critically ill paediatric patients

    PubMed Central

    2014-01-01

    Background Nosocomial candidaemia is associated with high mortality rates in critically ill paediatric patients; thus, the early detection and identification of the infectious agent is crucial for successful medical intervention. The PCR-based techniques have significantly increased the detection of Candida species in bloodstream infections. In this study, a multiplex nested PCR approach was developed for candidaemia detection in neonatal and paediatric intensive care patients. Methods DNA samples from the blood of 54 neonates and children hospitalised in intensive care units with suspected candidaemia were evaluated by multiplex nested PCR with specific primers designed to identify seven Candida species, and the results were compared with those obtained from blood cultures. Results The multiplex nested PCR had a detection limit of four Candida genomes/mL of blood for all Candida species. Blood cultures were positive in 14.8% of patients, whereas the multiplex nested PCR was positive in 24.0% of patients, including all culture-positive patients. The results obtained with the molecular technique were available within 24 hours, and the assay was able to identify Candida species with 100% of concordance with blood cultures. Additionally, the multiplex nested PCR detected dual candidaemia in three patients. Conclusions Our proposed PCR method may represent an effective tool for the detection and identification of Candida species in the context of candidaemia diagnosis in children, showing highly sensitive detection and the ability to identify the major species involved in this infection. PMID:25047415

  16. Universal primer PCR with DGGE for rapid detection of bacterial pathogens.

    PubMed

    Ji, Niannian; Peng, Bo; Wang, Guizhong; Wang, Sanying; Peng, Xuanxian

    2004-06-01

    A universal primer PCR (UPPCR) combined with denaturing gradient gel electrophoresis (DGGE) was evaluated as a method permitting the rapid detection of pathogens. The results show that this method is efficient at amplifying the conserved regions of bacterial 16S rRNA genes with universal primers and can detect causative bacterial pathogens rapidly. Six species of bacteria from fisheries (Pseudomonas fluorescens, Vibrio anguillarum, Aeromonas hydrophila, Vibrio fluvialis, Providencia rettgeri and Aeromonas sobria) were examined. Our results indicate that the approach we undertook can be adopted not only for axenic bacterial populations but also for mixed communities as well. Furthermore, we were able to achieve the rapid detection of multiple bacteria a single in sample. In addition, UPPCR-DGGE was shown to be better than previously reported UPPCR-single-stranded conformation polymorphism (SSCP)-based methods for the rapid detection of bacterial pathogens. PMID:15134888

  17. Detection and Differentiation of Listeria spp. by a Single Reaction Based on Multiplex PCR

    PubMed Central

    Bubert, Andreas; Hein, Inge; Rauch, Marcus; Lehner, Angelika; Yoon, ByoungSu; Goebel, Werner; Wagner, Martin

    1999-01-01

    The iap gene encodes the protein p60, which is common to all Listeria species. A previous comparison of the DNA sequences indicated conserved and species-specific gene portions. Based on these comparisons, a combination consisting of only five different primers that allows the specific detection and differentiation of Listeria species with a single multiplex PCR and subsequent gel analysis was selected. One primer was derived from the conserved 3′ end and is specific for all Listeria species; the other four primers are specific for Listeria monocytogenes, L. innocua, L. grayi, or the three grouped species L. ivanovii, L. seeligeri, and L. welshimeri, respectively. The PCR method, which also enables the simultaneous detection of L. monocytogenes and L. innocua, was evaluated against conventional biotyping with 200 food hygiene-relevant Listeria strains. The results indicated the superiority of this technique. Thus, this novel type of multiplex PCR may be useful for rapid Listeria species confirmation and for identification of Listeria species for strains isolated from different sources. PMID:10508109

  18. A Pentaplex PCR Assay for the Detection and Differentiation of Shigella Species

    PubMed Central

    Ojha, Suvash Chandra; Yean Yean, Chan; Ismail, Asma; Banga Singh, Kirnpal-Kaur

    2013-01-01

    The magnitude of shigellosis in developing countries is largely unknown because an affordable detection method is not available. Current laboratory diagnosis of Shigella spp. is laborious and time consuming and has low sensitivity. Hence, in the present study, a molecular-based diagnostic assay which amplifies simultaneously four specific genes to identify invC for Shigella genus, rfc for S. flexneri, wbgZ for S. sonnei, and rfpB for S. dysenteriae, as well as one internal control (ompA) gene, was developed in a single reaction to detect and differentiate Shigella spp. Validation with 120 Shigella strains and 37 non-Shigella strains yielded 100% specificity. The sensitivity of the PCR was 100 pg of genomic DNA, 5.4 × 104 CFU/ml, or approximately 120 CFU per reaction mixture of bacteria. The sensitivity of the pentaplex PCR assay was further improved following preincubation of the stool samples in Gram-negative broth. A preliminary study with 30 diarrhoeal specimens resulted in no cross-reaction with other non-Shigella strains tested. We conclude that the developed pentaplex PCR assay is robust and can provide information about the four target genes that are essential for the identification of the Shigella genus and the three Shigella species responsible for the majority of shigellosis cases. PMID:23509722

  19. Detection of Nicotiana DNA in Tobacco Products Using a Novel Multiplex Real-Time PCR Assay.

    PubMed

    Korchinski, Katie L; Land, Adrian D; Craft, David L; Brzezinski, Jennifer L

    2016-07-01

    Establishing that a product contains tobacco is a requirement for the U.S. Food and Drug Administration's regulation and/or prosecution of tobacco products. Therefore, a multiplex real-time PCR method was designed to determine if Nicotiana (tobacco) DNA is present in tobacco products. The PCR method simultaneously amplifies a 73 bp fragment of the cytochrome P450 monoxygenase CYP82E4 gene and 66 bp fragment in the nia-1 gene for nitrate reductase, which are detected using dual-labeled TaqMan probes. The assay is capable of detecting approximately 7.8 pg purified tobacco DNA, with a similar sensitivity for either gene target while incorporating an internal positive control (IPC). DNA was extracted from prepared tobacco products-including chewing tobacco, pipe tobacco, and snuff-or from the cut fill (no wrapper) of cigarettes and cigars. Of the 13 products analyzed, 12 were positive for both tobacco-specific markers and the IPC. DNA was also extracted from the fill of five varieties of herbal cigarettes, which were negative for both tobacco-specific gene targets and positive for the IPC. Our method expands on current assays by introducing a multiplex reaction, targeting two sequences in two different genes of interest, incorporating an IPC into the reaction, and lowering the LOD and LOQ while increasing the efficiency of the PCR. PMID:27143320

  20. Detection and analysis of diverse herpesviral species by consensus primer PCR.

    PubMed Central

    VanDevanter, D R; Warrener, P; Bennett, L; Schultz, E R; Coulter, S; Garber, R L; Rose, T M

    1996-01-01

    A consensus primer PCR method which amplifies a region of herpesviral DNA-directed DNA polymerase (EC 2.7.7.7) and which uses degenerate primers in a nested format was developed. Primers were designed to target sequences coding for highly conserved amino acid motifs covering a region of approximately 800 bp. The assay was applied to 22 species of herpesviruses (8 human and 14 animal viruses), with PCR products obtained for 21 of 22 viruses. In the process, 14 previously unreported amino acid-coding sequences from herpesviral DNA polymerases were obtained, including regions of human herpesviruses 7 and 8. The 50 to 60 amino acid-coding sequences recovered in the present study were determined to be unique to each viral species studied, with very little sequence variation between strains of a single species when studied. Template dilution studies in the presence of human carrier DNA demonstrated that six human herpesviruses (herpesviruses 1, 2, 3, 4, 5, and 6B) could be detected at levels at or below 100 genome equivalents per 100 ng of carrier DNA. These data suggest that consensus primer PCR targeted to herpesviral DNA polymerase may prove to be useful in the detection and identification of known herpesviruses in clinical samples and the initial characterization of new herpesviral genomes. PMID:8784566

  1. Rapid Quantitative Detection of Lactobacillus sakei in Meat and Fermented Sausages by Real-Time PCR

    PubMed Central

    Martín, Belén; Jofré, Anna; Garriga, Margarita; Pla, Maria; Aymerich, Teresa

    2006-01-01

    A quick and simple method for quantitative detection of Lactobacillus sakei in fermented sausages was successfully developed. It is based on Chelex-100-based DNA purification and real-time PCR enumeration using a TaqMan fluorescence probe. Primers and probes were designed in the L. sakei 16S-23S rRNA intergenic transcribed spacer region, and the assay was evaluated using L. sakei genomic DNA and an artificially inoculated sausage model. The detection limit of this technique was approximately 3 cells per reaction mixture using both purified DNA and the inoculated sausage model. The quantification limit was established at 30 cells per reaction mixture in both models. The assay was then applied to enumerate L. sakei in real samples, and the results were compared to the MRS agar count method followed by confirmation of the percentage of L. sakei colonies. The results obtained by real-time PCR were not statistically significantly different than those obtained by plate count on MRS agar (P > 0.05), showing a satisfactory agreement between both methods. Therefore, the real-time PCR assay developed can be considered a promising rapid alternative method for the quantification of L. sakei and evaluation of the implantation of starter strains of L. sakei in fermented sausages. PMID:16957227

  2. Real-Time PCR Assays for Detection of Bocavirus in Human Specimens

    PubMed Central

    Lu, Xiaoyan; Chittaganpitch, Malinee; Olsen, Sonja J.; Mackay, Ian M.; Sloots, Theo P.; Fry, Alicia M.; Erdman, Dean D.

    2006-01-01

    The recently discovered human bocavirus (HBoV) is the first member of the family Parvoviridae, genus Bocavirus, to be potentially associated with human disease. Several studies have identified HBoV in respiratory specimens from children with acute respiratory disease, but the full spectrum of clinical disease and the epidemiology of HBoV infection remain unclear. The availability of rapid and reliable molecular diagnostics would therefore aid future studies of this novel virus. To address this, we developed two sensitive and specific real-time TaqMan PCR assays that target the HBoV NS1 and NP-1 genes. Both assays could reproducibly detect 10 copies of a recombinant DNA plasmid containing a partial region of the HBoV genome, with a dynamic range of 8 log units (101 to 108 copies). Eight blinded clinical specimen extracts positive for HBoV by an independent PCR assay were positive by both real-time assays. Among 1,178 NP swabs collected from hospitalized pneumonia patients in Sa Kaeo Province, Thailand, 53 (4.5%) were reproducibly positive for HBoV by one or both targets. Our data confirm the possible association of HBoV infection with pneumonia and demonstrate the utility of these real-time PCR assays for HBoV detection. PMID:16954253

  3. Multiplex PCR detection of enterotoxin genes in Aeromonas spp. from suspect food samples in northern Taiwan.

    PubMed

    Chang, Yu-Chang; Wang, Jan-Yi; Selvam, Ammaiyappan; Kao, Shu-Chen; Yang, Shang-Shyng; Shih, Daniel Yang-Chih

    2008-10-01

    Aeromonads possess an array of virulence factors and are causative agents of a number of human infections. Among them, genes of one cytotoxic (Act) and two cytotonic (Alt, Ast) enterotoxins are implicated in a human diarrheal disease. A rapid, specific, simultaneous detection of these enterotoxin genes in suspected food poisoning samples is not yet reported. Hence, a multiplex PCR assay was designed to amplify the cytotoxic (act), heat-labile cytotonic (alt), and heat-stable cytotonic (ast) enterotoxin genes of aeromonads. The PCR assay was tested with 133 Aeromonas spp. isolated from suspect food poisoning samples and retail samples of poultry and fish from wet markets in and around Taipei, Northern Taiwan. The Aeromonas spp. isolates were divided into six genotypes based on absence or presence of one or more enterotoxin genes. Of these 133 isolates, Aeromonas caviae (52.5%) and Aeromonas hydrophila (43.4%) were the most frequently isolated species from food poisoning samples and retail samples, respectively. Among the species, A. hydrophila had a significantly higher proportion for harboring three enterotoxin genes than had the others, whereas Aeromonas encheleia, considered a nonpathogen, was found harboring three enterotoxin genes. The multiplex PCR assays are rapid and specific, and provide a useful tool for the detection and genotyping of enterotoxin genes of aeromonads. PMID:18939759

  4. A PCR-free fluorescence strategy for detecting telomerase activity via double amplification strategy.

    PubMed

    Zhang, Xiafei; Cheng, Rui; Shi, Zhilu; Jin, Yan

    2016-01-15

    As a universal tumor biomarker, research on the activity and inhibition of telomerase is of great importance for cancer diagnosis and therapy. Although the telomeric repeat amplification protocol (TRAP) has served as a powerful assay for detecting telomerase activity, its application has been significantly limited by amplification related errors and time-consuming procedure. To address the limitations of PCR-based protocol, a dual amplification fluorescence assay was developed for PCR-free detecting telomerase activity. Briefly, we designed an arch-structure DNA probe to specifically control strand displacement reaction and subsequent enzyme-aided amplification. Telomerase substrate (TS) primer was extended by telomerase to form long elongation products which contain several TTAGGG repeat units. So, one elongation product can release more than one trigger DNA (t-DNA) via strand displacement reaction to realize first amplification. Subsequently, t-DNA specifically opened molecular beacon (MB) to restore the fluorescence of MB. Meanwhile, t-DNA was recycled by the aid of nicking endonuclease to continuously open more and more MBs, leading to a second amplification. Owing to the double amplification strategy, the proposed method allowed the measurement of telomerase activity in crude cell extracts equivalent to 5 HeLa cells and 10 CCRF-CEM cells without PCR amplification. Besides, the influence of telomere-binding ligands on the telomerase activity demonstrated that the proposed method holds the potential to evaluate the inhibition efficiency of telomerase inhibitors. PMID:26299822

  5. A novel COLD-PCR/FMCA assay enhances the detection of low-abundance IDH1 mutations in gliomas.

    PubMed

    Pang, Brendan; Durso, Mary B; Hamilton, Ronald L; Nikiforova, Marina N

    2013-03-01

    Point mutations in isocitrate dehydrogenase 1 (IDH1) have been identified in many gliomas. The detection of IDH1 mutations becomes challenging on suboptimal glioma biopsies when a limited number of tumor cells is available for analysis. Coamplification at lower denaturing-polymerase chain reaction (COLD-PCR) is a PCR technique that deliberately lowers the denaturing cycle temperature to selectively favor amplification of mutant alleles, allowing for the sensitive detection of low-abundance mutations. We developed a novel COLD-PCR assay on the LightCycler platform (Roche, Applied Science, Indianapolis, IN), using post-PCR fluorescent melting curve analysis (FMCA) for the detection of mutant IDH1 with a detection limit of 1%. Thirty-five WHO grade I to IV gliomas and 9 non-neoplastic brain and spinal cord biopsies were analyzed with this technique and the results were compared with the conventional real-time PCR and the Sanger sequencing analysis. COLD-PCR/FMCA was able to detect the most common IDH1 R132H mutation and rare mutation types including R132H, R132C, R132L, R132S, and R132G mutations. Twenty-five glioma cases were positive for IDH1 mutations by COLD-PCR/FMCA, and 23 gliomas were positive by the conventional real-time PCR and Sanger sequencing. A pilocytic astrocytoma (PA I) and a glioblastoma multiforme (GBM IV) showed low-abundance IDH1 mutations detected by COLD-PCR/FMCA. The remaining 10 glioma and 9 non-neoplastic samples were negative by all the 3 methods. In summary, we report a novel COLD-PCR/FMCA method that provides rapid and sensitive detection of IDH1 mutations in formalin-fixed paraffin-embedded tissue and can be used in the clinical setting to assess the small brain biopsies. PMID:23370430

  6. Detection of Helicobacter pylori DNA in recurrent aphthous stomatitis tissue by PCR.

    PubMed

    Riggio, M P; Lennon, A; Wray, D

    2000-11-01

    Helicobacter pylori is recognised as being an aetiological agent of chronic active gastritis and peptic ulcer disease and has been associated with an increased risk of gastric cancer. The natural reservoir for H. pylori is unknown, although the oral cavity has been the focus of much attention in this respect. Given the histological similarities between gastric and oral ulceration, it seemed prudent to investigate a possible association between H. pylori and recurrent aphthous stomatitis (RAS). In this study, the potential involvement of H. pylori in the aetiology of RAS was investigated using the polymerase chain reaction (PCR). Biopsies from 28 RAS patients were analysed, in addition to 20 oral lichen planus (OLP) and 13 normal biopsies that were used as controls. Genomic DNA was extracted from biopsies, and confirmation of successful extraction of PCR-amplifiable DNA was achieved by carrying out PCR on each DNA sample with nested primers specific for the human beta-haemoglobin gene. PCR identification of H. pylori was carried out using a primer pair specific for the H. pylori 16S ribosomal RNA (rRNA) gene. Two rounds of PCR were carried out to amplify a 295-bp product, and the identity of amplified products was confirmed by DNA sequencing. H. pylori DNA was detected in 3 of 28 (11%) RAS samples but not in any of 20 OLP and 13 normal samples. These results do not support a definitive aetiological role for H. pylori in RAS, although the possibility that H. pylori may be involved in a small proportion of RAS cases cannot be excluded. PMID:11048967

  7. Highly Specific Detection of Five Exotic Quarantine Plant Viruses using RT-PCR.

    PubMed

    Choi, Hoseong; Cho, Won Kyong; Yu, Jisuk; Lee, Jong-Seung; Kim, Kook-Hyung

    2013-03-01

    To detect five plant viruses (Beet black scorch virus, Beet necrotic yellow vein virus, Eggplant mottled dwarf virus, Pelargonium zonate spot virus, and Rice yellow mottle virus) for quarantine purposes, we designed 15 RT-PCR primer sets. Primer design was based on the nucleotide sequence of the coat protein gene, which is highly conserved within species. All but one primer set successfully amplified the targets, and gradient PCRs indicated that the optimal temperature for the 14 useful primer sets was 51.9°C. Some primer sets worked well regardless of annealing temperature while others required a very specific annealing temperature. A primer specificity test using plant total RNAs and cDNAs of other plant virus-infected samples demonstrated that the designed primer sets were highly specific and generated reproducible results. The newly developed RT-PCR primer sets would be useful for quarantine inspections aimed at preventing the entry of exotic plant viruses into Korea. PMID:25288934

  8. Novel Polyomavirus Detected in the Feces of a Chimpanzee by Nested Broad-Spectrum PCR

    PubMed Central

    Johne, Reimar; Enderlein, Dirk; Nieper, Hermann; Müller, Hermann

    2005-01-01

    In order to screen for new polyomaviruses in samples derived from various animal species, degenerated PCR primer pairs were constructed. By using a nested PCR protocol, the sensitive detection of nine different polyomavirus genomes was demonstrated. The screening of field samples revealed the presence of a new polyomavirus, tentatively designated chimpanzee polyomavirus (ChPyV), in the feces of a juvenile chimpanzee (Pan troglodytes). Analysis of the region encoding the major capsid protein VP1 revealed a unique insertion in the EF loop of the protein and showed that ChPyV is a distinct virus related to the monkey polyomavirus B-lymphotropic polyomavirus and the human polyomavirus JC polyomavirus. PMID:15731285

  9. Novel polyomavirus detected in the feces of a chimpanzee by nested broad-spectrum PCR.

    PubMed

    Johne, Reimar; Enderlein, Dirk; Nieper, Hermann; Müller, Hermann

    2005-03-01

    In order to screen for new polyomaviruses in samples derived from various animal species, degenerated PCR primer pairs were constructed. By using a nested PCR protocol, the sensitive detection of nine different polyomavirus genomes was demonstrated. The screening of field samples revealed the presence of a new polyomavirus, tentatively designated chimpanzee polyomavirus (ChPyV), in the feces of a juvenile chimpanzee (Pan troglodytes). Analysis of the region encoding the major capsid protein VP1 revealed a unique insertion in the EF loop of the protein and showed that ChPyV is a distinct virus related to the monkey polyomavirus B-lymphotropic polyomavirus and the human polyomavirus JC polyomavirus. PMID:15731285

  10. Clinical Evaluation of COBAS TaqMan PCR for the Detection of Mycobacterium tuberculosis and M. avium Complex

    PubMed Central

    Ikegame, Satoshi; Sakoda, Yoritake; Fujino, Nao; Taguchi, Kazuhito; Kawasaki, Masayuki; Kajiki, Akira

    2012-01-01

    A retrospective observational study was performed to determine the sensitivity and limitation of PCR test for the detection of Mycobacterium tuberculosis and M. avium complex. We obtained clinical specimens collected from the respiratory tract, cultured M. tuberculosis or M. avium complex, and performed PCR analysis. A total of 299 samples (M. tuberculosis, 177; M. avium, 35; M. intracellulare, 87) were analyzed by COBAS TaqMan PCR from April 2007 to March 2011. The PCR positivity rates were 50–55%, 70–100%, 88–98%, and 100% in smear-negative, smear 1+, 2+, and 3+ groups, respectively. The PCR positivity of tuberculosis in smear 1+ was 80.6%, which was statistically significantly (P < 0.001) lower than that of smear 2+ (97.3%). From January 2005 to March 2007, we collected an additional 138 samples (M. tuberculosis, 74; M. avium, 21; M. intracellulare, 43), which were analyzed by COBAS Amplicor PCR. The PCR positivity rates obtained using COBAS TaqMan PCR and COBAS Amplicor PCR were not significantly different. The sensitivity of PCR test for mycobacteria is not sufficient in case of smear 1+. Careful consideration must be given to the interpretation of negative PCR test results in smear 1+, because smear-positive tuberculosis is the criterion for isolation. PMID:23029612

  11. Digital PCR methods improve detection sensitivity and measurement precision of low abundance mtDNA deletions

    PubMed Central

    Belmonte, Frances R.; Martin, James L.; Frescura, Kristin; Damas, Joana; Pereira, Filipe; Tarnopolsky, Mark A.; Kaufman, Brett A.

    2016-01-01

    Mitochondrial DNA (mtDNA) mutations are a common cause of primary mitochondrial disorders, and have also been implicated in a broad collection of conditions, including aging, neurodegeneration, and cancer. Prevalent among these pathogenic variants are mtDNA deletions, which show a strong bias for the loss of sequence in the major arc between, but not including, the heavy and light strand origins of replication. Because individual mtDNA deletions can accumulate focally, occur with multiple mixed breakpoints, and in the presence of normal mtDNA sequences, methods that detect broad-spectrum mutations with enhanced sensitivity and limited costs have both research and clinical applications. In this study, we evaluated semi-quantitative and digital PCR-based methods of mtDNA deletion detection using double-stranded reference templates or biological samples. Our aim was to describe key experimental assay parameters that will enable the analysis of low levels or small differences in mtDNA deletion load during disease progression, with limited false-positive detection. We determined that the digital PCR method significantly improved mtDNA deletion detection sensitivity through absolute quantitation, improved precision and reduced assay standard error. PMID:27122135

  12. Efficient detection of Alport syndrome COL4A5 mutations with multiplex genomic PCR-SSCP.

    PubMed

    Barker, D F; Denison, J C; Atkin, C L; Gregory, M C

    2001-01-15

    We have performed effective mutation screening of COL4A5 with a new method of direct, multiplex genomic amplification that employs a single buffer condition and PCR profile. Application of the method to a consecutive series of 46 United States patients with diverse indications of Alport syndrome resulted in detection of mutations in 31 cases and of five previously unreported polymorphisms. With a correction for the presence of cases that are not likely to be due to changes at the COL4A5 locus, the mutation detection sensitivity is greater than 79%. The test examines 52 segments, including the COL4A6/COL4A5 intergenic promoter region, all 51 of the previously recognized exons and two newly detected exons between exons 41 and 42 that encode an alternatively spliced mRNA segment. New genomic sequence information was generated and used to design primer pairs that span substantial intron sequences on each side of all 53 exons. For SSCP screening, 16 multiplex PCR combinations (15 4-plex and 1 3-plex) were used to provide complete, partially redundant coverage of the gene. The selected combinations allow clear resolution of products from each segment using various SSCP gel formulations. One of the 29 different mutations detected initially seemed to be a missense change in exon 32 but was found to cause exon skipping. Another missense variant may mark a novel functional site located in the collagenous domain. PMID:11223851

  13. Copro-PCR based detection of bovine schistosome infection in India.

    PubMed

    Lakshmanan, B; Devada, K; Joseph, S; Aravindakshan, T V; Sabu, L

    2016-01-01

    Schistosomosis and amphistomosis are the two economically important and widely prevalent snail-borne trematode infections in grazing cattle of southern India. Acute infections are symptomatically similar and difficult to detect by routine microscopy for eggs. The present study was directed towards the development of a copro-polymerase chain reaction (copro-PCR) for detection of bovine schistosome species, using custom-designed primers targeting 18S and 28S ribosomal RNA as well as mitochondrial DNA. The study demonstrated the enhanced diagnostic specificity of mitochondrial DNA markers over ribosomal RNA genes as genus-specific probes to detect schistosomes. We developed a sensitive PCR assay using primers designed from mitochondrial DNA sequences targeting the partial rrnl (16S rRNA), tCys (transfer RNA for cysteine) and partial rrnS (12S rRNA) genes of Schistosoma spindale to specifically detect schistosome infection from faecal samples of naturally infected bovines. The salient findings of the work also throw light on to the high similarity of the ribosomal RNA gene sequences of schistosomes with those of Gastrothylax crumenifer and Fischoederius elongatus, the most prevalent pouched amphistomes of the region. Further investigation has to be directed towards unravelling the complete gene sequences of 18S and 28S ribosomal RNA as well as mitochondrial DNA sequences of amphistome isolates from India. PMID:26693890

  14. Detection of aerosolized biological agents by immunoassay followed by autonomous PCR confirmation

    SciTech Connect

    Dzenitis, J M; Hindson, B J; McBride, M T; Makarewicz, A J; Henderer, B D; Sathyam, U S; Smith, S M; Gutierrez, D M; Metz, T R; Venkateswaran, K S; Colston, B W; Farrow, S W

    2003-12-15

    An Autonomous Pathogen Detection System (APDS) unit is an automated, podium-sized system that monitors the air for all three biological threat agents (bacteria, viruses, and toxins). The system has been developed under the auspices of the U. S. Department of Energy and Department of Homeland Security by the University of California, Lawrence Livermore National Laboratory (LLNL) to protect people in critical or high-traffic facilities and at special events. The system performs continuous aerosol collection, sample preparation, and multiplexed biological tests using advanced immunoassays as the primary screen. Over ten agents are assayed at once, and results are reported hourly. R&D work this year focused on incorporating polymerase chain-reaction (PCR) techniques for detecting DNA as confirmation of immunoassay positives. The primary objective of the Dugway testing was to demonstrate the APDS with immunoassay identification and PCR confirmation of bacteria. A secondary objective was to demonstrate immunoassay identification of a protein toxoid (denatured toxin) aerosol release. A total of 12 agent trials were conducted over 14 days of testing, for a total of four work weeks at Dugway. Both testing objectives were achieved with multiple releases and clear identifications. The APDS was shown to be effective for identifying aerosolized Bacillus anthracis, Yersinia pestis, Bacillus globigii, and botulinum toxoid. The two areas for improvement were operational as opposed to hardware-related. The first was slowing the PCR thermal cycling to achieve stronger signals, which was demonstrated during the later phases of testing. The second area is to improve the parameters for autonomous PCR triggering; this is one of the focuses of the upcoming year's work.

  15. Duplex Real-Time PCR for Rapid Simultaneous Detection of Batrachochytrium dendrobatidis and Batrachochytrium salamandrivorans in Amphibian Samples

    PubMed Central

    Pasmans, F.; Longcore, J. E.; Spitzen-van der Sluijs, A.; Vercammen, F.; Martel, A.

    2013-01-01

    Chytridiomycosis is a lethal fungal disease contributing to declines and extinctions of amphibian species worldwide. The currently used molecular screening tests for chytridiomycosis fail to detect the recently described species Batrachochytrium salamandrivorans. In this study, we present a duplex real-time PCR that allows the simultaneous detection of B. salamandrivorans and Batrachochytrium dendrobatidis. With B. dendrobatidis- and B. salamandrivorans-specific primers and probes, detection of the two pathogens in amphibian samples is possible, with a detection limit of 0.1 genomic equivalent of zoospores of both pathogens per PCR. The developed real-time PCR shows high degrees of specificity and sensitivity, high linear correlations (r2 > 0.995), and high amplification efficiencies (>94%) for B. dendrobatidis and B. salamandrivorans. In conclusion, the described duplex real-time PCR can be used to detect DNA of B. dendrobatidis and B. salamandrivorans with highly reproducible and reliable results. PMID:24108616

  16. Detection of PCR products from Mycobacterium avium subspecies Paratuberculosis using oligonucleotides containing multiple 2,4-dinitrophenyl reporter groups.

    PubMed

    Stevenson, K; Walker, C A; Grzybowski, J; Brown, T; Sharp, J M

    A pool of five oligonucleotides has been used to detect the pathogenic organism Mycobacterium avium subspecies paratuberculosis in PCR-amplified DNA from ruminants. The oligonucleotides were labelled at the 5'-end with three dinitrophenyl reporter groups and hybridised to the target DNA, which was fixed to a nylon membrane by ultraviolet irradiation. Colourimetric detection of the PCR product was carried out using an anti-DNP antibody conjugated to horseradish peroxidase or to alkaline phosphatase. Detection with alkaline phosphatase was more sensitive than with horseradish peroxidase but, in both cases, the PCR product could be easily detected. The DNP labelling system offers an economic and effective alternative to biotin, digoxigenin or fluorescein for the detection of PCR-amplified DNA. PMID:9346864

  17. Effective detection of human noroviruses in Hawaiian waters using enhanced RT-PCR methods.

    PubMed

    Tong, Hsin-I; Connell, Christina; Boehm, Alexandria B; Lu, Yuanan

    2011-11-15

    The current recreational water quality criteria using growth-based measurements of fecal indicator bacteria (FIB) concentration have their limitations for swimmer protection. To evaluate the possible use of enteric viruses as an improved indicator of human sewage contamination in recreational waters for enhanced health risk assessment, human norovirus (huNoV) was tested as a model in this study. To establish a highly sensitive protocol for effective huNoV detection in waters, 16 published and newly designed reverse transcription polymerase chain reaction (RT-PCR) primer pairs specific for huNoV genogroup I (GI) and genogroup II (GII) were comparatively evaluated side-by-side using single sources of huNoV RNA stock extracted from local clinical isolates. Under optimized conditions, these RT-PCR protocols shared a very different pattern of detection sensitivity for huNoV. The primer sets COG2F/COG2R and QNIF4/NV1LCR were determined to be the most sensitive ones for huNoV GII and GI, respectively, with up to 10(5)- and 10(6)-fold more sensitive as compared to other sets tested. These two sensitive protocols were validated by positive detection of huNoV in untreated and treated urban wastewater samples. In addition, these RT-PCR protocols enabled detection of the prevalence of huNoV in 5 (GI) and 10 (GII) of 16 recreational water samples collected around the island of O'ahu, which was confirmed by DNA sequencing and sequence analysis. Findings from this study support the possible use of enteric viral pathogens for environmental monitoring and argue the importance and essentiality for such monitoring activity to ensure a safe use of recreational waters. PMID:21945082

  18. Development of duplex real-time PCR for the detection of WSSV and PstDV1 in cultivated shrimp

    PubMed Central

    2014-01-01

    Background 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. Results 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. Conclusion The standardized duplex qPCR was shown to be a robust, highly sensitive, and feasible diagnostic tool for the

  19. Single Laboratory Comparison of Quantitative Real-Time PCR Assays for the Detection of Human Fecal Pollution

    EPA Science Inventory

    There are numerous quantitative real-time PCR (qPCR) methods available to detect and enumerate human fecal pollution in ambient waters. Each assay employs distinct primers and/or probes and many target different genes and microorganisms leading to potential variations in method ...

  20. Single Laboratory Comparison of Quantitative Real-Time PCR Assays for the Detection of Human Fecal Pollution - Poster

    EPA Science Inventory

    There are numerous quantitative real-time PCR (qPCR) methods available to detect and enumerate human fecal pollution in ambient waters. Each assay employs distinct primers and/or probes and many target different genes and microorganisms leading to potential variations in method p...

  1. Development of primers and probes for detection of citrus "Candidatus Liberibacter species" by real-time PCR

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Citrus huanglongbing (HLB) or citrus greening is the most threatening bacterial disease of Citrus spp. Three uncultured Candidatus Liberibacter spp. are usually detected by conventional PCR or real-time PCR using species specific primers and probes based on the 16S rRNA gene. Recent molecular analy...

  2. Determining the 95% limit of detection for waterborne pathogen analyses from primary concentration to qPCR

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The limit of detection (LOD) for qPCR-based analyses is not consistently defined or determined in studies on waterborne pathogens. Moreover, the LODs reported often reflect the qPCR assay rather than the entire sample process. Our objective was to develop a method to determine the 95% LOD (lowest co...

  3. PCR detection, identification to species level, and fingerprinting of Campylobacter jejuni and Campylobacter coli direct from diarrheic samples.

    PubMed Central

    Linton, D; Lawson, A J; Owen, R J; Stanley, J

    1997-01-01

    Three sets of primers were designed for PCR detection and differentiation of Campylobacter jejuni and Campylobacter coli. The first PCR assay was designed to coidentify C. jejuni and C. coli based on their 16S rRNA gene sequences. The second PCR assay, based on the hippuricase gene sequence, identified all tested reference strains of C. jejuni and also strains of that species which lack detectable hippuricase activity. The third PCR assay, based on the sequence of a cloned (putative) aspartokinase gene and the downstream open reading frame, identified all tested reference strains of C. coli. The assays will find immediate application in the rapid identification to species level of isolates. The assays combine with a protocol for purification of total DNA from fecal samples to allow reproducible PCR identification of campylobacters directly from stools. Of 20 clinical samples from which campylobacters had been cultured, we detected C. jejuni in 17, C. coli in 2, and coinfection of C. jejuni and Campylobacter hyointestinalis in 1. These results were concordant with culture and phenotypic identification to species level. Strain typing by PCR-restriction fragment length polymorphism of the flagellin (flaA) gene detected identical flaA types in fecal DNA and the corresponding campylobacter isolate. Twenty-five Campylobacter-negative stool samples gave no reaction with the PCR assays. These PCR assays can rapidly define the occurrence, species incidence, and flaA genotypes of enteropathogenic campylobacters. PMID:9316909

  4. Improved Specificity for Detection of Mycobacterium bovis in Fresh Tissues Using IS6110 Real-time PCR

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background: Culture of M. bovis from diagnostic specimens is the gold standard for bovine tuberculosis diagnostics in the US. Detection of M. bovis by PCR in tissue homogenates may provide a simple, rapid method to complement diagnostic culture. A significant impediment to PCR based assays on tissue...

  5. Detection of Salmonellae in Chicken Feces by a Combination of Tetrathionate Broth Enrichment, Capillary PCR, and Capillary Gel Electrophoresis

    PubMed Central

    Carli, K. Tayfun; Unal, Can Bora; Caner, Vildan; Eyigor, Aysegul

    2001-01-01

    This report describes a rapid detection procedure for salmonellae from chicken feces by the combination of tetrathionate primary enrichment (preenrichment [PE])-bacterial lysis-capillary PCR and capillary gel electrophoresis. Pure Salmonella enterica serovar Enteritidis 64K was reisolated and detected by capillary PCR after buffered peptone water and nutrient broth, tetrathionate broth base Hajna (TTBH), and tetrathionate broth (TTB) preenrichments. When the same culture was mixed with intestinal homogenate, bacteriological reisolation and capillary PCR detection was achieved only by TTBH and TTB preenrichments. Capillary gel electrophoresis revealed that a Salmonella genus-specific 281-bp PCR product was detected when Salmonella strains but not non-Salmonella strains were tested. The detection limit of capillary PCR with whole-cell DNA extracted from pure Salmonella enterica serovars Enteritidis 64K, Typhimurium LT2-CIP60-62, and Gallinarum 64K was 3, 3, and 9 CFU ml−1, respectively. The detection limit of capillary PCR from whole-cell DNA extracted from intestinal homogenate artificially contaminated with the same three strains was 3, 3, and 7 CFU ml−1, respectively. We compared the results of the capillary PCR and bacteriological examination from the natural samples. Thirty-five of 53 naturally contaminated samples produced a specific PCR product. In 9 of the 35 PCR-positive samples, Salmonella could not be detected bacteriologically either by PE or a primary and delayed secondary enrichment (DSE) combination. In the 18 PCR-negative samples, 4 samples were found to harbor Salmonella by both PE and DSE and 14 samples were positive after DSE. Fifty-three additional intestinal homogenate samples, which were negative by their PE and DSE in bacteriological examination, were found to be also negative by their PCRs. The total time required to detect Salmonella with the capillary PCR method we used was approximately 20 h. If samples are from clinically diseased

  6. Development of a dry reagent-based triplex PCR for the detection of toxigenic and non-toxigenic Vibrio cholerae.

    PubMed

    Chua, Ang Lim; Elina, Husni Tan; Lim, Boon Huat; Yean, Chan Yean; Ravichandran, Manickam; Lalitha, Pattabhiraman

    2011-04-01

    Vibrio cholerae has caused severe outbreaks of cholera worldwide with thousands of recorded deaths annually. Molecular diagnosis for cholera has become increasingly important for rapid detection of cholera as the conventional methods are time-consuming and labour intensive. However, traditional PCR tests still require cold-chain transportation and storage as well as trained personnel to perform, which makes them user-unfriendly. The aim of this study was to develop a thermostabilized triplex PCR test for cholera which is in a ready-to-use form and requires no cold chain. The PCR test specifically detects both toxigenic and non-toxigenic strains of V. cholerae based on the cholera toxin A (ctxA) and outer-membrane lipoprotein (lolB) genes. The thermostabilized triplex PCR also incorporates an internal amplification control that helps to check for PCR inhibitors in samples. PCR reagents and the specific primers were lyophilized into a pellet form in the presence of trehalose, which acts as an enzyme stabilizer. The triplex PCR was validated with 174 bacteria-spiked stool specimens and was found to be 100 % sensitive and specific. The stability of the thermostabilized PCR was evaluated using the Q10 method and it was found to be stable for approximately 7 months at 24 °C. The limit of detection of the thermostabilized triplex PCR assay was 2×10(4) c.f.u. at the bacterial cell level and 100 pg DNA at the genomic DNA level, comparable to conventional PCR methods. In conclusion, a rapid thermostabilized triplex PCR assay was developed for detecting toxigenic and non-toxigenic V. cholerae which requires minimal pipetting steps and is cold chain-free. PMID:21183596

  7. Detection and discrimination of Loa loa, Mansonella perstans and Wuchereria bancrofti by PCR-RFLP and nested-PCR of ribosomal DNA ITS1 region.

    PubMed

    Jiménez, Maribel; González, Luis Miguel; Carranza, Cristina; Bailo, Begoña; Pérez-Ayala, Ana; Muro, Antonio; Pérez-Arellano, José Luis; Gárate, Teresa

    2011-01-01

    The ribosomal deoxyribonucleic acid (DNA) internal transcribed spacer region (ITS1) of two filarial nematodes, Loa loa and Mansonella perstans, was amplified and further sequenced to develop an species-specific polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) protocol for the differentiation of both species from Wuchereria bancrofti, three filarial nematodes with blood circulating microfilariae. The ITS1-PCR product digested with the restriction endonuclease Ase I generated an specific diagnostic pattern for each of the three species. Moreover, three new specific nested-PCRs, targeting the ITS1 region, for differential detection of L. loa, M. perstans and W. bancrofti were developed and used when the ITS1-PCR products were insufficient for the Ase I enzymatic digestion. These filarial species-specific molecular protocols were evaluated in forty blood samples from African adult immigrants attending in the Hospital Insular of Gran Canaria, Canarias, Spain. PMID:20599994

  8. Validation of real-time PCR assays for bioforensic detection of model plant pathogens.

    PubMed

    James, Mindy; Blagden, Trenna; Moncrief, Ian; Burans, James P; Schneider, Katherine; Fletcher, Jacqueline

    2014-03-01

    The U.S. agricultural sector is vulnerable to intentionally introduced microbial threats because of its wide and open distribution and economic importance. To investigate such events, forensically valid assays for plant pathogen detection are needed. In this work, real-time PCR assays were developed for three model plant pathogens: Pseudomonas syringae pathovar tomato, Xylella fastidiosa, and Wheat streak mosaic virus. Validation included determination of the linearity and range, limit of detection, sensitivity, specificity, and exclusivity of each assay. Additionally, positive control plasmids, distinguishable from native signature by restriction enzyme digestion, were developed to support forensic application of the assays. Each assay displayed linear amplification of target nucleic acid, detected 100 fg or less of target nucleic acid, and was specific to its target pathogen. Results obtained with these model pathogens provide the framework for development and validation of similar assays for other plant pathogens of high consequence. PMID:24261870

  9. Multiplex PCR assay for the detection of five meat species forbidden in Islamic foods.

    PubMed

    Ali, Md Eaqub; Razzak, Md Abdur; Hamid, Sharifah Bee Abd; Rahman, Md Mahfujur; Amin, Md Al; Rashid, Nur Raifana Abd; Asing

    2015-06-15

    Food falsification has direct impact on public health, religious faith, fair-trades and wildlife. For the first time, here we described a multiplex polymerase chain reaction assay for the accurate identification of five meat species forbidden in Islamic foods in a single assay platform. Five pairs of species-specific primers were designed targeting mitochondrial ND5, ATPase 6, and cytochrome b genes to amplify 172, 163, 141, 129 and 108 bp DNA fragments from cat, dog, pig, monkey and rat meats, respectively. All PCR products were identified in gel-images and electrochromatograms obtained from Experion Bioanalyzer. Species-specificity checking against 15 important meat and fish and 5 plant species detected no cross-species amplification. Screening of target species in model and commercial meatballs reflected its application to detect target species in process foods. The assay was tested to detect 0.01-0.02 ng DNA under raw states and 1% suspected meats in meatball formulation. PMID:25660879

  10. Detection of hepatitis B virus genotypic resistance mutations by coamplification at lower denaturation temperature-PCR coupled with sanger sequencing.

    PubMed

    Liu, Can; Lin, Jinpiao; Chen, Huijuan; Shang, Hongyan; Jiang, Ling; Chen, Jing; Ye, Yang; Yang, Bin; Ou, Qishui

    2014-08-01

    Mutations in the reverse transcriptase (rt) region of the DNA polymerase gene are the primary cause of hepatitis B virus (HBV) drug resistance. In this study, we established a novel method that couples coamplification at lower denaturation temperature (COLD)-PCR and Sanger sequencing, and we applied it to the detection of known and unknown HBV mutations. Primers were designed based on the common mutations in the HBV rt sequence at positions 180 to 215. The critical denaturation temperature (Tc) was established as a denaturing temperature for both fast and full COLD-PCR procedures. For single mutations, when a melting temperature (Tm)-reducing mutation occurred (e.g., C-G → T-A), the sensitivities of fast and full COLD-PCR for mutant detection were 1% and 2%, respectively; when the mutation caused no change in Tm (e.g., C-G → G-C) or raised Tm (e.g., T-A → C-G), only full COLD-PCR improved the sensitivity for mutant detection (2%). For combination mutations, the sensitivities of both full and fast COLD-PCR were increased to 0.5%. The limits of detection for fast and full COLD-PCR were 50 IU/ml and 100 IU/ml, respectively. In 30 chronic hepatitis B (CHB) cases, no mutations were detected by conventional PCR, whereas 18 mutations were successfully detected by COLD-PCR, including low-prevalence mutations (<10%), as confirmed by ultradeep pyrosequencing. In conclusion, COLD-PCR provides a highly sensitive, simple, inexpensive, and practical tool for significantly improving amplification efficacy and detecting low-level mutations in clinical CHB cases. PMID:24899029

  11. Application of tetraplex PCR for detection of Vibrio cholerae, V. parahaemolyticus, V. vulnificus and V. mimicus in cockle.

    PubMed

    Senachai, Pachara; Chomvarin, Chariya; Namwat, Wises; Wongboot, Warawan; Wongwajana, Suwin; Tangkanakul, Waraluk

    2013-03-01

    A tetraplex PCR method was developed for simultaneous detection of Vibrio cholerae, V. parahaemolyticus, V. vulnificus and V. mimicus in cockle samples in comparison with conventional culture method. Specific primers targeting ompW of V. cholerae, tl of V. parahaemolyticus, hsp60 of V. vulnificus and sodB of V. mimicus were employed in the same PCR. Detection limit of the tetraplex PCR assay was 104 cfu/ml (400 cfu/PCR reaction) for pure cultures of all four species of Vibrio. In Vibrio spiked cockle samples, the limit of detection after 6 hours enrichment in alkaline peptone water was 1 cfu/10 g of cockle tissue for three Vibrio spp, except for V. mimicus that was 102 cfu/10 g of cockle tissue. When the tetraplex PCR and culture methods were applied to 100 cockle samples, V. parahaemolyticus, V. vulnificus, V. cholerae and V. mimicus were detected in 100, 98, 80 and 9% of the samples by tetraplex PCR and in 76, 42, 0 and 0% by the culture method, respectively. This developed tetraplex PCR method should be suitable for simultaneous and rapid detection of Vibrio species in food samples and for food safety assessment. PMID:23691635

  12. Detection of feline coronavirus RNA in feces, tissues, and body fluids of naturally infected cats by reverse transcriptase PCR.

    PubMed Central

    Herrewegh, A A; de Groot, R J; Cepica, A; Egberink, H F; Horzinek, M C; Rottier, P J

    1995-01-01

    A nested reverse transcriptase PCR (RT-nPCR) was developed for the detection of feline coronavirus (FCoV) RNA in the feces, tissues, and body fluids of infected cats. The RT-nPCR was targeted to the highly conserved 3'-untranslated region of the viral genome and will detect most, if not all, feline coronaviruses in the field. With the RT-nPCR, FCoV RNA was detected in plasma samples from experimentally infected cats as early as 2 days postinoculation. FCoV RNA was also detected in serum, plasma, or ascitic fluid samples from 14 of 18 cats (78%) with naturally occurring feline infectious peritonitis (FIP). The use of RT-PCR for FIP diagnosis is limited because of the occurrence of apparently healthy FCoV carriers. These asymptomatic cats shed the virus in the feces and, in a number of cases, also had detectable virus in the plasma. Because of the nature of FCoV infections, our RT-PCR assay with plasma or serum cannot be used to establish a definite diagnosis of FIP. However, this assay does provide a new means to identify asymptomatic FCoV carriers. As such, RT-nPCR will be of use to screen cats before their introduction into FCoV-free catteries. Moreover, this assay provides an important tool to study the epidemiology of FCoV. PMID:7751377

  13. Laboratory Tests of Multiplex Detection of PCR Amplicons Using the Luminex 100 Flow Analyzer

    SciTech Connect

    Venkateswaran, K.S.; Nasarabadi, S.; Langlois, R.G.

    2000-05-05

    Lawrence Livermore National Laboratory (LLNL) demonstrated the power of flow cytometry in detecting the biological agents simulants at JFT III. LLNL pioneered in the development of advanced nucleic acid analyzer (ANM) for portable real time identification. Recent advances in flow cytometry provide a means for multiplexed nucleic acid detection and immunoassay of pathogenic microorganisms. We are presently developing multiplexed immunoassays for the simultaneous detection of different simulants. Our goal is to build an integrated instrument for both nucleic acid analysis and immuno detection. In this study we evaluated the Luminex LX 100 for concurrent identification of more than one PCR amplified product. ANAA has real-time Taqman fluorescent detection capability for rapid identification of field samples. However, its multiplexing ability is limited by the combination of available fluorescent labels. Hence integration of ANAA with flow cytometry can give the rapidity of ANAA amplification and the multiplex capability of flow cytometry. Multiplexed flow cytometric analysis is made possible using a set of fluorescent latex microsphere that are individually identified by their red and infrared fluorescence. A green fluorochrome is used as the assay signal. Methods were developed for the identification of specific nucleic acid sequences from Bacillus globigii (Bg), Bacillus thuringensis (Bt) and Erwinia herbicola (Eh). Detection sensitivity using different reporter fluorochromes was tested with the LX 100, and also different assay formats were evaluated for their suitability for rapid testing. A blind laboratory trial was carried out December 22-27, 1999 to evaluate bead assays for multiplex identification of Bg and Bt PCR products. This report summarizes the assay development, fluorochrome comparisons, and the results of the blind trial conducted at LLNL for the laboratory evaluation of the LX 100 flow analyzer.

  14. Rapid Detection of Ceratocystis platani Inoculum by Quantitative Real-Time PCR Assay

    PubMed Central

    Ghelardini, Luisa; Belbahri, Lassaâd; Quartier, Marion; Santini, Alberto

    2013-01-01

    Ceratocystis platani is the causal agent of canker stain of plane trees, a lethal disease able to kill mature trees in one or two successive growing seasons. The pathogen is a quarantine organism and has a negative impact on anthropogenic and natural populations of plane trees. Contaminated sawdust produced during pruning and sanitation fellings can contribute to disease spread. The goal of this study was to design a rapid, real-time quantitative PCR assay to detect a C. platani airborne inoculum. Airborne inoculum traps (AITs) were placed in an urban setting in the city of Florence, Italy, where the disease was present. Primers and TaqMan minor groove binder (MGB) probes were designed to target cerato-platanin (CP) and internal transcribed spacer 2 (ITS2) genes. The detection limits of the assay were 0.05 pg/μl and 2 fg/μl of fungal DNA for CP and ITS, respectively. Pathogen detection directly from AITs demonstrated specificity and high sensitivity for C. platani, detecting DNA concentrations as low as 1.2 × 10−2 to 1.4 × 10−2 pg/μl, corresponding to ∼10 conidia per ml. Airborne inoculum traps were able to detect the C. platani inoculum within 200 m of the closest symptomatic infected plane tree. The combination of airborne trapping and real-time quantitative PCR assay provides a rapid and sensitive method for the specific detection of a C. platani inoculum. This technique may be used to identify the period of highest risk of pathogen spread in a site, thus helping disease management. PMID:23811499

  15. 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. PMID:16461651

  16. PCR versus Southern blot detection of somatic mosaicism in fragile X syndrome

    SciTech Connect

    Mueller, O.T.; Amar, M.J.A.; Gallardo, L.A.; Kousseff, B.G.

    1994-09-01

    The incidence of somatic mosaicism in males with fragile X syndrome has been reported to be as high as 17% of all clinically affected males. Mosaic cases usually do not show the cytogenetic fragile site at Xq27.3 and generally are fully affected according to the clinical criteria, although some subjects show somewhat milder symptoms. Detection of mosaicism relies on the identification of multiple distinct sizes of the CGG size anomaly in the 5{prime} untranslated exon of the FMR-1 gene. Some alleles identified are of a premutation size with trinucleotides numbering 50 to 80 X CGG. In addition, there is a greatly expanded allele with well over 200 copies of the CGG repeat detected. We screened 314 subjects for fragile X syndrome over a three year period. Cases were routinely screened by Southern blotting using the StB12.3 probe as well as by polymerase chain reaction. The PCR amplification products were electrophoresed in agarose, blotted and hybridized with a (CGG){sub 5} oligonucleotide followed by chemiluminescent detection. Seventeen males and 16 females were identified with a CGG expansion, including two males with premutations with repeat sizes of between 50 and 100 CGG trinucleotides and three cases exhibiting somatic mosaicism. The mosaic cases had both a premutation-sized allele and one or more expanded alleles of over 250 CGG copies. The mosaic cases were usually undetected with Southern blotting but easily identified with this PCR protocol. The relative proportion of the expanded allele as determined by scanning densitometry were 70%, 35%, and 5% in the three cases. All three cases were cytogenetically negative. The clinical severity of the mosaic cases was variable, with symptoms ranging from severe MR with most of the physical stigmata to mild learning disability. In our experience, Southern blotting allows more accurate sizing of the expanded allele; however, PCR is essential to identify cases that exhibit mosaicism.

  17. Detection and quantification of Histomonas meleagridis by real-time PCR targeting single copy genes.

    PubMed

    Hussain, Imtiaz; Jaskulska, Barbara; Hess, Michael; Bilic, Ivana

    2015-09-15

    Histomonas meleagridis, a protozoan parasite that can infect gallinaceous birds, affects mainly the liver and caeca of infected birds. As a consequence of the recent ban of chemotherapeuticals in Europe and the USA, histomonosis gained somewhat more attention due to its re-emergence and the fact that there is no effective treatment available. Therefore, special attention is now also given towards the diagnosis and the control of the disease. In the actual study we report the development of highly specific and sensitive real-time PCR methods for detection and quantification of the parasite, based on two protein coding genes, Fe-hydrogenase (FeHYD) and rpb1. Both genes seem to be in a single copy in H. meleagridis as shown by southern blotting and absolute quantification using real-time PCRs on samples containing a known amount of the parasite. The real-time PCR assays based on FeHYD and rpb1 genes were found to be an efficient method for the quantification and detection of H. meleagridis in in vitro grown cultures, tissues of infected birds and in faecal samples. Both real-time PCRs were able to detect up to a single cell in in vitro cultures of H. meleagridis and in fecal samples spiked with H. meleagridis. Finally, qPCR assays were shown to be highly specific for H. meleagridis as samples containing either of the two H. meleagridis genotypes were positive, whereas samples containing other protozoa such as Tetratrichomonas gallinarum, Trichomonas gallinae, Simplicimonas sp., Tritrichomonas sp., Parahistomonas wenrichi, Dientamoebidae sp. and Blastocystis sp. were all negative. PMID:26319200

  18. Effective PCR-based detection of Naegleria fowleri from cultured sample and PAM-developed mouse.

    PubMed

    Kang, Heekyoung; Seong, Gi-Sang; Sohn, Hae-Jin; Kim, Jong-Hyun; Lee, Sang-Eun; Park, Mi Yeoun; Lee, Won-Ja; Shin, Ho-Joon

    2015-10-01

    Increasing numbers of Primary Amoebic Meningoencephalitis (PAM) cases due to Naegleria fowleri are becoming a serious issue in subtropical and tropical countries as a Neglected Tropical Disease (NTD). To establish a rapid and effective diagnostic tool, a PCR-based detection technique was developed based on previous PCR methods. Four kinds of primer pairs, Nfa1, Nae3, Nf-ITS, and Naegl, were employed in the cultured amoebic trophozoites and a mouse with PAM experimentally developed by N. fowleri inoculation (PAM-mouse). For the extraction of genomic DNA from N. fowleri trophozoites (1×10(6)), simple boiling with 10μl of PBS (pH 7.4) at 100°C for 30min was found to be the most rapid and efficient procedure, allowing amplification of 2.5×10(2) trophozoites using the Nfa-1 primer. The primers Nfa1 and Nae3 amplified only N. fowleri DNA, whereas the ITS primer detected N. fowleri and N. gruberi DNA. Using the PAM-mouse brain tissue, the Nfa1 primer was able to amplify the N. fowleri DNA 4 days post infection with 1ng/μl of genomic DNA being detectable. Using the PAM-mouse CSF, amplification of the N. fowleri DNA with the Nae3 primer was possible 5 days post infection showing a better performance than the Nfa1 primer at day 6. PMID:26322498

  19. Gene-Based Detection of Microorganisms in Environmental Samples Using PCR

    NASA Technical Reports Server (NTRS)

    Glass, John I.; Lefkowitz, Elliot J.; Cassell, Gail H.; Wechser, Mark; Taylor, Theresa B.; Albin, Michael; Paszko-Kolva, Christine; Roman, Monsi C.

    1997-01-01

    Contaminating microorganisms pose a serious potential risk to the crew's well being and water system integrity aboard the International Space Station (ISS). We are developing a gene-based microbial monitor that functions by replicating specific segments of DNA as much as 10(exp 12) x. Thus a single molecule of DNA can be replicated to detectable levels, and the kinetics of that molecule's accumulation can be used to determine the original concentration of specific microorganisms in a sample. Referred to as the polymerase chain reaction (PCR), this enzymatic amplification of specific segments of the DNA or RNA from contaminating microbes offers the promise of rapid, sensitive, quantitative detection and identification of bacteria, fungi, viruses, and parasites. We envision a small instrument capable of assaying an ISS water sample for 48 different microbes in a 24 hour period. We will report on both the developments in the chemistry necessary for the PCR assays to detect microbial contaminants in ISS water, and on progress towards the miniaturization and automation of the instrumentation.

  20. Real-time PCR assay for detection of a new simulant for poxvirus biothreat agents.

    PubMed

    Garnier, Laurence; Gaudin, Jean-Christophe; Bensadoun, Paul; Rebillat, Isabelle; Morel, Yannick

    2009-03-01

    Research and financial efforts spent on biodefense technologies highlight the current concern for biothreat event preparedness. Nonhazardous but relevant "simulant" microorganisms are typically used to simplify technological developments, testing, and staff training. The bacteriophage MS2, a small RNA virus, is classically used as the reference simulant for biothreat viruses within the biodefense community. However, variola virus, considered a major threat, displays very different features (size, envelope, and double-stranded DNA genome). The size parameter is critical for aerosol sampling, detection, and protection/filtration technologies. Therefore, a panel of relevant simulants should be used to cover the diversity of biothreat agents. Thus, we investigated a new virus model, the Cydia pomonella granulovirus (baculovirus), which is currently used as a biopesticide. It displays a size similar to that of poxviruses, is enveloped, and contains double-stranded DNA. To provide a molecular tool to detect and quantify this model virus, we developed an assay based on real-time PCR, with a limit of detection ranging from roughly 10 to a few tens of target copies per microl according to the sample matrix. The specificity of the assay against a large panel of potential cross-reactive microorganisms was checked, and the suitability of the assay for environmental samples, especially aerosol studies, was determined. In conclusion, we suggest that our PCR assay allows Cydia pomonella granulovirus to be used as a simulant for poxviruses. This assay may also be useful for environmental or crop treatment studies. PMID:19168659

  1. Multiplex real-time PCR SYBR Green for detection and typing of group III Clostridium botulinum.

    PubMed

    Anniballi, Fabrizio; Auricchio, Bruna; Delibato, Elisabetta; Antonacci, Monia; De Medici, Dario; Fenicia, Lucia

    2012-01-27

    Clostridium botulinum type C and type D belonging to the group III organisms, are mainly responsible for animal botulism outbreaks. Clinical signs alone are often insufficient to make a diagnosis of botulism and a laboratory confirmation is required. Laboratory confirmation can be performed by demonstrating the presence of botulinum neurotoxins in serum, gastrointestinal contents, liver, wound of sick or dead animals, or by demonstrating the presence of C. botulinum in gastrointestinal contents, liver, and wound. Demonstration of spores in gastrointestinal contents or tissue of animals with clinical signs indicative of botulism reinforces the clinical diagnosis. With the aim of detecting and typing C. botulinum group III organisms, a multiplex real-time PCR SYBR Green was developed and in-house validated. Selectivity, limit of detection, relative accuracy, relative specificity, relative sensitivity, and repeatability of the method were investigated. The multiplex real-time PCR SYBR green used showed a 100% selectivity, 100% relative accuracy, 100% relative specificity, 100% relative sensitivity and a limit of detection of 277 and 580 DNA copies for C. botulinum type C and C. botulinum type D, respectively. The method reported here represents a suitable tool for laboratory diagnosis of type C and D botulism and for testing a large number of samples collected during the animal botulism surveillance and prevention activities. PMID:21890285

  2. A short target real-time RT-PCR assay for detection of pestiviruses infecting cattle.

    PubMed

    La Rocca, S A; Sandvik, T

    2009-10-01

    A rapid single step real-time duplex TaqMan RT-PCR was developed for detection of bovine viral diarrhoea virus (BVDV)-1, BVDV-2 and border disease virus (BDV). Based on alignment of available and newly generated partial 5'-UTR nucleotide sequences, one forward and two reverse primers were designed, which amplify a 104bp PCR product. Two TaqMan probes labelled with different fluorochromes were designed to detect BVDV-1/BVDV-2 and BDVs, respectively. The assay was able to detect a selection of strains and isolates that represent the genetic diversity of these three viruses, with an analytical sensitivity that corresponded to 3.6, 48 and 4.8 TCID(50) of BVDV-1, BVDV-2 and BDV, respectively. With an overall cycling time of around 70 min, the assay allows rapid diagnosis and efficient use of modern thermocycling machines. Although developed principally for the diagnosis of BVD, the assay should be equally useful for diagnosis of BD in sheep. PMID:19523981

  3. Coupling electrochemical response of a DNA biosensor with PCR for Neisseria gonorrhoeae detection.

    PubMed

    Verma, Rachna; Sood, Seema; Singh, Renu; Sumana, Gajjala; Bala, Manju; Sharma, Vinod K; Samantaray, Jyotish C; Pandey, Ravindra M; Malhotra, Bansi D

    2014-01-01

    Early diagnosis of gonococcal infections is important with regard to a patient's health and stage of infection. In this context, we report the development of an opa-gene-based electrochemical DNA biosensor for detection of Neisseria gonorrhoeae by monitoring redox peak of methylene blue indicator. The fabricated biosensor has been shown to be highly sensitive and specific when evaluated with complementary, non-complementary, and 1-base mismatch DNA sequences and polymerase chain reaction (PCR) amplified products (amplicons) of standard strain of N. gonorrhoeae (ATCC49226). The biosensor has been further evaluated using amplicons of known positive and negative clinical samples, and cut-off for positives has been determined using receiver operating characteristic curve. The sensitivity (SN), specificity (SP), positive predictive value, and negative predictive value of the biosensor have been found to be 96.2%, 88.2%, 92.6%, and 93.8%, respectively. We conclude that the combination of PCR amplification with electrochemical detection shows distinct advantage of high SN and increased SP for gonococcal detection. PMID:24207077

  4. Specific detection of Aspergillus parasiticus in wheat flour using a highly sensitive PCR assay.

    PubMed

    Sardiñas, Noelia; Vázquez, Covadonga; Gil-Serna, Jessica; González-Jaen, M Teresa; Patiño, Belén

    2010-06-01

    Aspergillus parasiticus is one of the most important aflatoxin-producing species that contaminates foodstuffs and beverages for human consumption. In this work, a specific and highly sensitive PCR protocol was developed to detect A. parasiticus using primers designed on the multicopy internal transcribed region of the rDNA unit (ITS1-5.8S-ITS2 rDNA). The assay proved to be highly specific for A. parasiticus when tested on a wide range of related and other fungal species commonly found in commodities, and allowing discrimination from the closely related A. flavus. Accuracy of detection and quantification by conventional PCR were tested with genomic DNA obtained from wheat flour artificially contaminated with spore suspensions of known concentrations. Spore concentrations equal or higher than 10(6) spore/g could be detected by the assay directly without prior incubation of the samples. The assay described is suitable for incorporation in routine analyses at critical points of the food chain within HACCP strategies. PMID:20486001

  5. Collaborative evaluation of a method for the detection of Norwalk virus in shellfish tissues by PCR.

    PubMed Central

    Atmar, R L; Neill, F H; Woodley, C M; Manger, R; Fout, G S; Burkhardt, W; Leja, L; McGovern, E R; Le Guyader, F; Metcalf, T G; Estes, M K

    1996-01-01

    A multicenter, collaborative trial was performed to evaluate the reliability and reproducibility of a previously described method for the detection of Norwalk virus in shellfish tissues with the PCR (R.L. Atmar, F. H. Neill, J. L. Romalde, F. Le Guyader, C. M. Woodley, T. G. Metcalf, and M. K. Estes, Appl. Environ. Microbiol. 61:3014-3018, 1995). Virus was added to the stomachs and hepatopancreatic tissues of oysters or hard-shell clams in the control laboratory, the samples were shipped to the participating laboratories, and viral nucleic acids were extracted and then detected by reverse transcription-PCR. The sensitivity and specificity of the assay were 85 and 91%, respectively, when results were determined by visual inspection of ethidium bromide-stained agarose gels; the test sensitivity and specificity improved to 87 and 100%, respectively, after confirmation by hybridization with a digoxigenin-labeled, virus-specific probe. We have demonstrated that this method can be implemented successfully by several laboratories to detect Norwalk virus in shellfish tissues. PMID:8572702

  6. Detection of four important Eimeria species by multiplex PCR in a single assay.

    PubMed

    You, Myung-Jo

    2014-06-01

    The oocysts of some of the recognized species of chicken coccidiosis are difficult to distinguish morphologically. Diagnostic laboratories are increasingly utilizing DNA-based technologies for the specific identification of Eimeria species. This study reports a multiplex polymerase chain reaction (PCR) assay based on internal transcribed spacer-1 (ITS-1) for the simultaneous diagnosis of the Eimeria tenella, Eimeria acervulina, Eimeria maxima, and Eimeria necatrix species, which infect domestic fowl. Primer pairs specific to each species were designed in order to generate a ladder of amplification products ranging from 20 to 25 bp, and a common optimum annealing temperature for these species was determined to be 52.5 °C. Sensitivity tests were performed for each species, showing a detection threshold of 1-5 pg. All the species were amplified homogeneously, and a homogenous band ladder was observed, indicating that the assay permitted the simultaneous detection of all the species in a single-tube reaction. In the phylogenic study, there was a clear species clustering, which was irrespective of geographical location, for all the ITS-1 sequences used. This multiplex PCR assay represents a rapid and potential cost-effective diagnostic method for the detection of some key Eimeria species that infect domestic fowl. PMID:24495953

  7. Detection of African horse sickness virus in Culicoides imicola pools using RT-qPCR.

    PubMed

    de Waal, Tania; Liebenberg, Danica; Venter, Gert J; Mienie, Charlotte Ms; van Hamburg, Huib

    2016-06-01

    African horse sickness (AHS) is an infectious, non-contagious arthropod-borne disease of equids, caused by the African horse sickness virus (AHSV), an orbivirus of the Reoviridae family. It is endemic in sub-Saharan Africa and thought to be the most lethal viral disease of horses. This study focused on detection of AHSV in Culicoides imicola (Diptera: Ceratopogonidae) pools by the application of a RT-qPCR. Midges were fed on AHSV-infected blood. A single blood-engorged female was allocated to pools of unfed nulliparous female midges. Pool sizes varied from 1 to 200. RNA was extracted and prepared for RT-qPCR. The virus was successfully detected and the optimal pool size for the limit of detection of the virus was determined at a range between 1 to 25. Results from this investigation highlight the need for a standardized protocol for AHSV investigation in Culicoides midges especially for comparison among different studies and for the determination of infection rate. PMID:27232141

  8. Ultra–sensitive droplet digital PCR for detecting a low–prevalence somatic GNAQ mutation in Sturge–Weber syndrome

    PubMed Central

    Uchiyama, Yuri; Nakashima, Mitsuko; Watanabe, Satoshi; Miyajima, Masakazu; Taguri, Masataka; Miyatake, Satoko; Miyake, Noriko; Saitsu, Hirotomo; Mishima, Hiroyuki; Kinoshita, Akira; Arai, Hajime; Yoshiura, Ko–ichiro; Matsumoto, Naomichi

    2016-01-01

    Droplet digital PCR (ddPCR), a method for measuring target nucleic acid sequence quantity, is useful for determining somatic mutation rates using TaqMan probes. In this study, the detection limit of copy numbers of test DNA by ddPCR was determined based on Poisson distribution. Peptide nucleic acid (PNA), which strongly hybridises to target lesions, can inhibit target amplification by PCR. Therefore, by combination of PCR with PNA and ddPCR (PNA–ddPCR), the detection limit could be lowered. We reanalysed a somatic GNAQ mutation (c.548G > A) in patients with Sturge–Weber syndrome (SWS) using ddPCR and PNA–ddPCR. Importantly, among three patients previously found to be mutation negative by next–generation sequencing, two patients had the GNAQ mutation with a mutant allele frequency of less than 1%. Furthermore, we were able to find the same mutation in blood leukocyte or saliva DNA derived from four out of 40 SWS patients. Vascular anomalies and blood leukocytes originate from endothelial cells and haemangioblasts, respectively, which are both of mesodermal origin. Therefore, blood leukocytes may harbour the GNAQ mutation, depending on the time when the somatic mutation is acquired. These data suggest the possibility of diagnosis using blood DNA in some patients with SWS. PMID:26957145

  9. Ultra-sensitive droplet digital PCR for detecting a low-prevalence somatic GNAQ mutation in Sturge-Weber syndrome.

    PubMed

    Uchiyama, Yuri; Nakashima, Mitsuko; Watanabe, Satoshi; Miyajima, Masakazu; Taguri, Masataka; Miyatake, Satoko; Miyake, Noriko; Saitsu, Hirotomo; Mishima, Hiroyuki; Kinoshita, Akira; Arai, Hajime; Yoshiura, Ko-ichiro; Matsumoto, Naomichi

    2016-01-01

    Droplet digital PCR (ddPCR), a method for measuring target nucleic acid sequence quantity, is useful for determining somatic mutation rates using TaqMan probes. In this study, the detection limit of copy numbers of test DNA by ddPCR was determined based on Poisson distribution. Peptide nucleic acid (PNA), which strongly hybridises to target lesions, can inhibit target amplification by PCR. Therefore, by combination of PCR with PNA and ddPCR (PNA-ddPCR), the detection limit could be lowered. We reanalysed a somatic GNAQ mutation (c.548G > A) in patients with Sturge-Weber syndrome (SWS) using ddPCR and PNA-ddPCR. Importantly, among three patients previously found to be mutation negative by next-generation sequencing, two patients had the GNAQ mutation with a mutant allele frequency of less than 1%. Furthermore, we were able to find the same mutation in blood leukocyte or saliva DNA derived from four out of 40 SWS patients. Vascular anomalies and blood leukocytes originate from endothelial cells and haemangioblasts, respectively, which are both of mesodermal origin. Therefore, blood leukocytes may harbour the GNAQ mutation, depending on the time when the somatic mutation is acquired. These data suggest the possibility of diagnosis using blood DNA in some patients with SWS. PMID:26957145

  10. Simultaneous PCR detection of Haemophilus ducreyi, Treponema pallidum, and herpes simplex virus types 1 and 2 from genital ulcers.

    PubMed Central

    Orle, K A; Gates, C A; Martin, D H; Body, B A; Weiss, J B

    1996-01-01

    A multiplex PCR (M-PCR) assay with colorimetric detection was devised for the simultaneous amplification of DNA targets from Haemophilus ducreyi, Treponema pallidum, and herpes simplex virus (HSV) types 1 and 2. By using target-specific oligonucleotides in a microwell format, 298 genital ulcer swab specimens collected in New Orleans during three intervals from 1992 through 1994 were evaluated. The results of the M-PCR assay were compared with the results of dark-field microscopy and H. ducreyi culture on two different culture media. HSV culture results were available for 99 specimens collected during the third interval. Confirmatory PCR assays targeting different gene sequences for each of the three organisms were used to validate the M-PCR results. Specimens were resolved as positive for the determination of sensitivity if the reference diagnostic test was positive or if the results of both the M-PCR and the confirmatory PCR were positive. The resolved sensitivities of M-PCR for HSV, H. ducreyi, and T. pallidum were 100, 98.4, and 91%, respectively. The resolved sensitivities of HSV culture, H. ducreyi culture, and dark-field microscopy were 71.8, 74.2, and 81%, respectively. These results indicate that the M-PCR assay is more sensitive than standard diagnostic tests for the detection of HSV, H. ducreyi, and T. pallidum from genital ulcers. PMID:8748271

  11. Detection of Dientamoeba fragilis in animal faeces using species specific real time PCR assay.

    PubMed

    Chan, Douglas; Barratt, Joel; Roberts, Tamalee; Phillips, Owen; Šlapeta, Jan; Ryan, Una; Marriott, Deborah; Harkness, John; Ellis, John; Stark, Damien

    2016-08-30

    Dientamoeba fragilis is a potentially pathogenic, enteric, protozoan parasite with a worldwide distribution. While clinical case reports and prevalence studies appear regularly in the scientific literature, little attention has been paid to this parasite's biology, life cycle, host range, and possible transmission routes. Overall, these aspects of Dientamoeba biology remain poorly understood at best. In this study, a total of 420 animal samples, collected from Australia, were surveyed for the presence of Dientamoeba fragilis using PCR. Several PCR assays were evaluated for sensitivity and specificity. Two previously published PCR methods demonstrated cross reactivity with other trichomonads commonly found in animal samples. Only one assay exhibited excellent specificity. Using this assay D. fragilis was detected from one dog and one cat sample. This is the first report of D. fragilis from these animals and highlights the role companion animals may play in D. fragilis transmission. This study demonstrated that some published D. fragilis molecular assays cross react with other closely related trichomonads and consequently are not suitable for animal prevalence studies. PMID:27523936

  12. Detection of wild animals as carriers of Leptospira by PCR in the Pantanal biome, Brazil.

    PubMed

    Vieira, Anahi S; Narduche, Lorena; Martins, Gabriel; Schabib Péres, Igor A H F; Zimmermann, Namor P; Juliano, Raquel S; Pellegrin, Aiesca O; Lilenbaum, Walter

    2016-11-01

    Leptospiral infection is widespread in wildlife. In this context, wild ecosystems in tropical countries hold a vast biodiversity, including several species that may act as potential reservoirs of leptospires. The Pantanal biome presents highly favorable environmental conditions for the occurrence of leptospirosis, such as high temperatures, constant flooding, and high biodiversity. The purpose of this study was to detect wild animals as carriers of Leptospira sp. using direct methods (PCR and culture) in the Pantanal biome, Brazil. A total of 35 animals were studied, namely Cerdocyon thous, Nasua nasua, Ozotoceros bezoarticus, and Sus scrofa species. Blood for serology (MAT) and urine for bacteriological culturing and PCR was sampled. The most prevalent serogroups were Javanica and Djasiman. Additionally, 40.6% of these animals presented PCR positive reactions. Seroreactivity associated with the high frequency of leptospiral carriers among the different studied species suggests a high level of exposure of the studied animals to pathogenic Leptospira strains. Our results are still limited and the actual role of the studied animals in the epidemiology of leptospirosis in the Pantanal region remains to be elucidated. PMID:27496621

  13. Detection of five Shiga toxin-producing Escherichia coli genes with multiplex PCR.

    PubMed

    Son, Insook; Binet, Rachel; Maounounen-Laasri, Anna; Lin, Andrew; Hammack, Thomas S; Kase, Julie A

    2014-06-01

    Escherichia coli serogroup O157 is the pathogen most commonly associated with foodborne disease outbreaks, but epidemiological studies suggest that non-O157 Shiga toxin-producing E. coli (STEC) is a major player as well. The ten most clinically relevant STECs belong to serogroups O26, O103, O111, O145, O157, O91, O113, O128, O45, and O121; but emerging strains, such as O104:H4 that was identified with the 2011 German outbreak, could become more prevalent in the future. A 75-min conventional multiplex PCR assay, IS-5P, targeting the four virulence factors stx1, stx2, eae, and ehxA plus the O157:H7-specific +93 uidA single nucleotide polymorphism was developed to better assess the potential pathogenicity of STEC isolates. All 212 STEC DNAs showed one to five amplification products, while the non-E. coli DNA did not react to this multiplex PCR assay. Enrichment broths obtained from baby spinach, alfalfa sprouts, and cilantro artificially inoculated with O26, O103, and O121 STECs reacted positively to the multiplex assay. Unlike the current FDA BAM 5P PCR, designed for the specific detection of O157:H7, IS-5P will identify potentially harmful O157:H7 and non-O157 STECs so they can be removed from the nation's food supply. PMID:24549195

  14. Babesia canis vogeli: a novel PCR for its detection in dogs in Australia.

    PubMed

    Martin, Anthony R; Dunstan, Richard H; Roberts, Timothy K; Brown, Graeme K

    2006-01-01

    Babesia canis vogeli is known to cause disease in dogs in Australia, and the rapid detection of various subspecies would enable effective treatment and management. A 21 bp oligonucleotide, "Bab-f" was proposed for the production of larger PCR products with high species specificity that would enable effective sequence analyses to yield subspecies identification. The new forward primer when paired with a previously reported "Babesia common" reverse primer generated a 394 bp product which was successfully amplified and provided subspecies differentiation by sequence analyses. Specificity and sensitivity were reported at 100% on a cohort of 55 dogs. PMID:16256109

  15. 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. PMID:27036222

  16. High Frequency of Detection by PCR of Viral Nucleic Acid in The Blood of Infants Presenting with Clinical Myocarditis.

    PubMed

    Simpson, Kathleen E; Storch, Gregory A; Lee, Caroline K; Ward, Kent E; Danon, Saar; Simon, Catherine M; Delaney, Jeffrey W; Tong, Alan; Canter, Charles E

    2016-02-01

    Specific viruses are associated with pediatric myocarditis, but the prevalence of viral DNAemia detected by blood polymerase chain reaction (PCR) is unknown. We evaluated the prevalence of known cardiotropic viruses (enterovirus, adenovirus, human herpesvirus 6, and parvovirus B19) in children with clinical myocarditis (n = 21). Results were compared to pediatric controls with similar viral PCR testing. The majority of positive PCR (89 %) was noted in children ≤12 months of age at diagnosis compared to older children. Infant myocarditis patients (8/10) had increased the prevalence of PCR positivity compared to infant pediatric controls (4/114) (p < 0.0001). Other than age, patient characteristics at diagnosis were similar between PCR-positive and PCR-negative patients. Both PCR-negative myocarditis infants had clinical recovery at follow-up. Of the PCR-positive myocarditis infants, 4 had clinical recovery, 2 developed chronic cardiomyopathy, 1 underwent heart transplant, and 1 died. Infants with clinical myocarditis have a high rate of blood viral positivity, which is higher compared to older children with myocarditis and healthy infant controls. Age-related differences in PCR positivity may be due to differences in host and/or virus characteristics. Our findings suggest that viral blood PCR may be a useful diagnostic tool and identify patients who would potentially benefit from virus-specific therapy. PMID:26499513

  17. Low copy target detection by Droplet Digital PCR through application of a novel open access bioinformatic pipeline, ‘definetherain’

    PubMed Central

    Jones, Mathew; Williams, James; Gärtner, Kathleen; Phillips, Rodney; Hurst, Jacob; Frater, John

    2014-01-01

    Droplet Digital PCR (ddPCR) represents a new and alternative platform to conventional quantitative-PCR (qPCR) for the quantitation of DNA templates. However, the proposed improvement in sensitivity and reproducibility offered by ddPCR is not yet fully proven, partly because the delineation between positive and negative responses is not always clear. Data are presented demonstrating the sensitivity of the ddPCR system to both reagent concentrations and choice of cut-off for defining positive and negative results. By implementing k-nearest clustering, cut-offs are produced that improve the accuracy of ddPCR where target DNA is present at low copy numbers, a key application of ddPCR. This approach is applied to human albumin and HIV-1 proviral DNA ddPCR quantitative protocols. This tool is coded in JavaScript and has been made available for free in a web browser at http://www.definetherain.org.uk. Optimisation of the analyses of raw ddPCR data using ‘definetherain’ indicates that low target number detection can be improved by its implementation. Further application to patient samples will help define the clinical utility of this approach. PMID:24598230

  18. Low copy target detection by Droplet Digital PCR through application of a novel open access bioinformatic pipeline, 'definetherain'.

    PubMed

    Jones, Mathew; Williams, James; Gärtner, Kathleen; Phillips, Rodney; Hurst, Jacob; Frater, John

    2014-06-01

    Droplet Digital PCR (ddPCR) represents a new and alternative platform to conventional quantitative-PCR (qPCR) for the quantitation of DNA templates. However, the proposed improvement in sensitivity and reproducibility offered by ddPCR is not yet fully proven, partly because the delineation between positive and negative responses is not always clear. Data are presented demonstrating the sensitivity of the ddPCR system to both reagent concentrations and choice of cut-off for defining positive and negative results. By implementing k-nearest clustering, cut-offs are produced that improve the accuracy of ddPCR where target DNA is present at low copy numbers, a key application of ddPCR. This approach is applied to human albumin and HIV-1 proviral DNA ddPCR quantitative protocols. This tool is coded in JavaScript and has been made available for free in a web browser at http://www.definetherain.org.uk. Optimisation of the analyses of raw ddPCR data using 'definetherain' indicates that low target number detection can be improved by its implementation. Further application to patient samples will help define the clinical utility of this approach. PMID:24598230

  19. Species markers for equine strongyles detected in intergenic rDNA by PCR-RFLP.

    PubMed

    Gasser, R B; Stevenson, L A; Chilton, N B; Nansen, P; Bucknell, D G; Beveridge, I

    1996-10-01

    Five species of equine strongyle belonging to the subfamily Strongylinae (Strongylus edentatus, S. equinus, S. vulgaris, Oesophagodontus robustus and Triodontophorus serratus) and 11 species belonging to the subfamily Cyathostominae (Poteriostomum imparidentatum, P. ratzii, Cylicocyclus insignis, Cc. leptostomus, Cc. nassatus, Cylicostephanus calicatus, Cs. longibursatus, Cs. goldi, Cyathostomum catinatum, Cy. labiatum and Cy. pateratum) were characterized using a polymerase chain reaction-linked restriction fragment length polymorphism technique (PCR-RFLP). Internal transcribed spacer ribosomal DNA was amplified from genomic DNA by polymerase chain reaction (PCR) using conserved primers, digested separately with six restriction endonucleases (AluI, BfaI, CfoI, Hae III, VSpI and XbaI) and the fragments separated by agarose gel electrophoresis. The PCR products of the three Strongylus species were approx. 90-100 bp smaller in size compared with those of the other 13 species. The PCR-RFLP analysis of the rDNA region spanning the first and second internal transcribed spacers plus the 5.85 rDNA gene (ITS+) produced characteristic patterns for each of the 16 species examined, and no variation in RFLP patterns was detected within the species Cy. catinatum, where multiple isolates were analysed. The study demonstrates that the internal transcribed spacer sequences provide genetic markers for the species identification of a range of equine strongyles. These markers will be of use for the identification of egg and larval stages, where morphological characters alone are unreliable. The results also indicate that the spacer sequences will be of use to study the systematics of equine strongyles. PMID:8910892

  20. Impact of HIV Infection Status on Interpretation of Quantitative PCR for Detection of Pneumocystis jirovecii

    PubMed Central

    Louis, M.; Guitard, J.; Jodar, M.; Ancelle, T.; Magne, D.; Lascols, O.

    2015-01-01

    Quantitative PCR (qPCR) is now a key diagnostic tool for Pneumocystis pneumonia. However, cutoffs to distinguish between infected and colonized patients according to their HIV status have not yet been determined. According to clinical, radiological, and biological data, we retrospectively classified bronchoalveolar lavage (BAL) samples subjected to qPCR over a 3-year period into four categories, i.e., definite PCP, probable PCP, Pneumocystis colonization, and no infection. Fungal burden was then analyzed according to the HIV status of the patients. Among 1,212 episodes of pneumonia screened in immunocompromised patients, 52 and 27 HIV-positive patients were diagnosed with a definite and probable PCP, whereas 4 and 22 HIV-negative patients had definite and probable PCP, respectively. Among patients with definite or a probable PCP, HIV-negative patients had a significantly lower burden than HIV-positive patients (P < 10−4). In both groups, the median fungal burden was significantly higher in patients with definite PCP than in colonized patients. A single cutoff at 1.5 × 104 copies/ml allowed to differentiate colonized and infected HIV-positive patients with 100% sensitivity and specificity. In HIV-negative patients, cutoff values of 2.87 × 104 and 3.39 × 103 copies/ml resulted in 100% specificity and sensitivity, respectively. Using cutoffs determined for the whole population would have led us to set aside the diagnosis of PCP in 9 HIV-negative patients with definite or probable PCP. qPCR appeared to be the most sensitive test to detect Pneumocystis in BAL samples. However, because of lower inocula in HIV-negative patients, different cutoffs must be used according to the HIV status to differentiate between colonized and infected patients. PMID:26468505

  1. Impact of HIV Infection Status on Interpretation of Quantitative PCR for Detection of Pneumocystis jirovecii.

    PubMed

    Louis, M; Guitard, J; Jodar, M; Ancelle, T; Magne, D; Lascols, O; Hennequin, C

    2015-12-01

    Quantitative PCR (qPCR) is now a key diagnostic tool for Pneumocystis pneumonia. However, cutoffs to distinguish between infected and colonized patients according to their HIV status have not yet been determined. According to clinical, radiological, and biological data, we retrospectively classified bronchoalveolar lavage (BAL) samples subjected to qPCR over a 3-year period into four categories, i.e., definite PCP, probable PCP, Pneumocystis colonization, and no infection. Fungal burden was then analyzed according to the HIV status of the patients. Among 1,212 episodes of pneumonia screened in immunocompromised patients, 52 and 27 HIV-positive patients were diagnosed with a definite and probable PCP, whereas 4 and 22 HIV-negative patients had definite and probable PCP, respectively. Among patients with definite or a probable PCP, HIV-negative patients had a significantly lower burden than HIV-positive patients (P < 10(-4)). In both groups, the median fungal burden was significantly higher in patients with definite PCP than in colonized patients. A single cutoff at 1.5 × 10(4) copies/ml allowed to differentiate colonized and infected HIV-positive patients with 100% sensitivity and specificity. In HIV-negative patients, cutoff values of 2.87 × 10(4) and 3.39 × 10(3) copies/ml resulted in 100% specificity and sensitivity, respectively. Using cutoffs determined for the whole population would have led us to set aside the diagnosis of PCP in 9 HIV-negative patients with definite or probable PCP. qPCR appeared to be the most sensitive test to detect Pneumocystis in BAL samples. However, because of lower inocula in HIV-negative patients, different cutoffs must be used according to the HIV status to differentiate between colonized and infected patients. PMID:26468505

  2. Detection of Human Herpesvirus 6 and Varicella-Zoster Virus in Tear Fluid of Patients with Bell's Palsy by PCR

    PubMed Central

    Pitkäranta, A.; Piiparinen, H.; Mannonen, L.; Vesaluoma, M.; Vaheri, A.

    2000-01-01

    Human herpesvirus 6 DNA was detected by PCR in the tear fluid of 7 (35%) of 20 patients with Bell's palsy and of 1 (5%) of 20 healthy controls. Varicella-zoster virus was detected by PCR in the tear fluid of 2 of 20 Bell's palsy patients but in none of the tear fluids from 20 healthy controls. These findings suggest an association between human herpesviruses and Bell's palsy. PMID:10878079

  3. Detection of maternal DNA in umbilical cord plasma by fluorescent PCR amplification of short tandem repeat sequences.

    PubMed

    Bauer, M; Orescovic, I; Schoell, W M; Bianchi, D W; Pertl, B

    2001-09-01

    Recently, maternal DNA was detected in umbilical cord blood using PCR amplification of minisatellite sequences. The presence of maternal DNA was demonstrated in 1% to 100% of umbilical cord blood samples. The objective of this study was to determine the frequency of cord blood contamination by maternal genetic material. We used fluorescent PCR amplification of highly polymorphic short tandem repeat (STR) markers to detect maternal DNA in umbilical cord plasma. PMID:11708473

  4. Egg intensity and freeze-thawing of fecal samples affect sensitivity of Echinococcus multilocularis detection by PCR.

    PubMed

    Klein, C; Liccioli, S; Massolo, A

    2014-10-01

    Echinococcus multilocularis is one of the most relevant zoonotic parasites with about 18,000 human cases per year. Its detection in wild host is crucial for disease prevention. The present study aimed to determine factors affecting the sensitivity of E. multilocularis detection by PCR using DNA extracted from fecal samples of coyotes (Canis latrans). Fecal samples were screened for the presence of Taeniidae eggs through centrifugation and sedimentation. DNA was extracted from fecal samples with and without prior freeze-thawing of the sample and then subjected to PCR targeting a mitochondrial gene (nad1) and a multi-loci microsatellite marker (EmsB). The presence of PCR inhibitors was determined through internal amplification control. Subjecting the sample to repeated freeze-thaw cycles significantly increased the sensitivity of the PCR by 20%. Likewise, egg intensity had a significant effect on PCR, an effect which was more pronounced for samples not subjected to freeze-thawing. Two or more eggs per gram of feces significantly increased the odds for a positive PCR outcome. The presence of PCR inhibitors had no effect on PCR in samples subjected to freeze-thaw cycles, whereas in samples not subjected to freeze-thaw cycles, the presence of PCR inhibitors was associated with a 0.78 lower odds ratio of positive PCR outcome. Targeting a nuclear versus a mitochondrial gene did not have a significant effect on the sensitivity of PCR. We recommend freeze-thawing samples prior to DNA extraction to become a standard procedure for E. multilocularis detection in canid feces. PMID:25082017

  5. Genetic detection of Babesia bigemina from Mongolian cattle using apical membrane antigen-1 gene-based PCR assay.

    PubMed

    Sivakumar, Thillaiampalam; Altangerel, Khukhuu; Battsetseg, Badgar; Battur, Banzragch; Aboulaila, Mahmoud; Munkhjargal, Tserendorj; Yoshinari, Takeshi; Yokoyama, Naoaki; Igarashi, Ikuo

    2012-06-01

    We developed a new nested PCR (nPCR) assay based on the Babesia bigemina apical membrane antigen-1 (AMA-1) gene sequence for parasite-specific detection. The primers were designed to amplify 738-bp and 211-bp fragments of the AMA-1 gene by primary and nested PCRs, respectively. The assay was proven to be specific for the B. bigemina, whereas the previously established SpeI-AvaI nPCR assay amplified not only the target fragment of B. bigemina but also a homologous one from Babesia ovata. The AMA-1 nPCR assay was also evaluated using field DNA samples extracted from 266 bovine blood samples collected from Mongolia in 2010. In a comparative evaluation, 90 (33.8%) and 25 (9.4%) of the blood samples showed positive reactions for B. bigemina by the SpeI-AvaI nPCR and AMA-1 nPCR assays, respectively. The sequencing analysis of the nPCR products confirmed that the AMA-1 nPCR method had specifically detected the target B. bigemina DNA. However, 4 different kinds of sequences were determined among the SpeI-AvaI nPCR amplicons. Two of them were derived from B. bigemina and B. ovata, while the origins of the others were unknown. In the current study, the presence of B. bigemina was clearly demonstrated among Mongolian cattle populations by the current nPCR assay for the first time. Furthermore, our findings also indicate that the AMA-1 nPCR assay may be a useful diagnostic tool for the specific detection of B. bigemina. PMID:22284301

  6. In situ detection of the Clostridium botulinum type C1 toxin gene in wetland sediments with a nested PCR assay

    USGS Publications Warehouse

    Williamson, J.L.; Rocke, T.E.; Aiken, Judd M.

    1999-01-01

    A nested PCR was developed for detection of the Clostridium botulinum type C1 toxin gene in sediments collected from wetlands where avian botulism outbreaks had or had not occurred. The C1 toxin gene was detected in 16 of 18 sites, demonstrating both the ubiquitous distribution of C. botulinum type C in wetland sediments and the sensitivity of the detection assay.

  7. Development of a multiplex PCR detection kit for Salmonella spp., Listeria monocytogenes, and Escherichia coli O157:H7

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A multiplex PCR method was developed for simultaneous detection of Salmonella spp., Listeria monocytogenes, and Escherichia coli O157:H7 in food samples, and the detection sensitivity for this method was 103 CFU/ml for each pathogen. When this method was used for the detection of each of pathogens ...

  8. Detection of Brucella spp. in bottlenose dolphins Tursiops truncatus by a real-time PCR using blowhole swabs.

    PubMed

    Wu, Qingzhong; Conway, Jessica; Phillips, Kristen M; Stolen, Megan; Durden, Wendy N; Fauquier, Deborah; McFee, Wayne E; Schwacke, Lori

    2016-08-01

    Blowhole swabs are a simple and non-invasive method for collecting samples from cetaceans and can be used for screening large numbers of animals in the field. This study reports a real-time PCR assay for the detection of Brucella spp. using blowhole swab samples from bottlenose dolphins Tursiops truncatus stranded in the coastal region of Virginia, South Carolina and northern Florida, USA, between 2013 and 2015. We used real-time PCR results on lung samples from the same dolphins in order to estimate the relative sensitivity and specificity of real-time PCR of blowhole swabs. Brucella DNA was detected in lung tissue of 22% (18/81) and in blowhole swabs of 21% (17/81) of the sampled dolphins. The relative sensitivity and specificity of real-time PCR on blowhole swabs as compared to the real-time PCR on lung samples was 94% (17/18) and 100% (63/63), respectively. These results indicate that real-time PCR on blowhole swabs may be used as a non-invasive test for rapid detection of Brucella spp. in the respiratory tract of dolphins. To our knowledge, this is the first report on the use of blowhole swabs for detection of bacterial pathogens by real-time PCR in bottlenose dolphins. PMID:27503920

  9. Detection of MPLW515L/K Mutations and Determination of Allele Frequencies with a Single-Tube PCR Assay

    PubMed Central

    Takei, Hiraku; Morishita, Soji; Araki, Marito; Edahiro, Yoko; Sunami, Yoshitaka; Hironaka, Yumi; Noda, Naohiro; Sekiguchi, Yuji; Tsuneda, Satoshi; Ohsaka, Akimichi; Komatsu, Norio

    2014-01-01

    A gain-of-function mutation in the myeloproliferative leukemia virus (MPL) gene, which encodes the thrombopoietin receptor, has been identified in patients with essential thrombocythemia and primary myelofibrosis, subgroups of classic myeloproliferative neoplasms (MPNs). The presence of MPL gene mutations is a critical diagnostic criterion for these diseases. Here, we developed a rapid, simple, and cost-effective method of detecting two major MPL mutations, MPLW515L/K, in a single PCR assay; we termed this method DARMS (dual amplification refractory mutation system)-PCR. DARMS-PCR is designed to produce three different PCR products corresponding to MPLW515L, MPLW515K, and all MPL alleles. The amplicons are later detected and quantified using a capillary sequencer to determine the relative frequencies of the mutant and wild-type alleles. Applying DARMS-PCR to human specimens, we successfully identified MPL mutations in MPN patients, with the exception of patients bearing mutant allele frequencies below the detection limit (5%) of this method. The MPL mutant allele frequencies determined using DARMS-PCR correlated strongly with the values determined using deep sequencing. Thus, we demonstrated the potential of DARMS-PCR to detect MPL mutations and determine the allele frequencies in a timely and cost-effective manner. PMID:25144224

  10. Specific Detection of Viable Legionella Cells by Combined Use of Photoactivated Ethidium Monoazide and PCR/Real-Time PCR▿

    PubMed Central

    Chang, Bin; Sugiyama, Kanji; Taguri, Toshitsugu; Amemura-Maekawa, Junko; Kura, Fumiaki; Watanabe, Haruo

    2009-01-01

    Legionella organisms are prevalent in manmade water systems and cause legionellosis in humans. A rapid detection method for viable Legionella cells combining ethidium monoazide (EMA) and PCR/real-time PCR was assessed. EMA could specifically intercalate and cleave the genomic DNA of heat- and chlorine-treated dead Legionella cells. The EMA-PCR assay clearly showed an amplified fragment specific for Legionella DNA from viable cells, but it could not do so for DNA from dead cells. The number of EMA-treated dead Legionella cells estimated by real-time PCR exhibited a 104- to 105-fold decrease compared to the number of dead Legionella cells without EMA treatment. Conversely, no significant difference in the numbers of EMA-treated and untreated viable Legionella cells was detected by the real-time PCR assay. The combined assay was also confirmed to be useful for specific detection of culturable Legionella cells from water samples obtained from spas. Therefore, the combined use of EMA and PCR/real-time PCR detects viable Legionella cells rapidly and specifically and may be useful in environmental surveillance for Legionella. PMID:18978073

  11. Development of a real-time quantitative RT-PCR to detect REV contamination in live vaccine.

    PubMed

    Luan, Huaibiao; Wang, Yixin; Li, Yang; Cui, Zhizhong; Chang, Shuang; Zhao, Peng

    2016-09-01

    Based on the published Avian reticuloendotheliosis virus (REV) whole genome sequence, primers and TaqMan probes were designed and synthesized, and the TaqMan probe fluorescence real-time quantitative RT-PCR (qRT-PCR) method for detecting the REV pol gene was established by optimizing the reaction conditions. Sensitivity analysis showed that the qRT-PCR method had a sensitivity that was 1,000-fold higher than conventional PCR. Additionally, no amplification signals were obtained when we attempted to detect DNA or cDNA of ALV-A/B/J, MDV, CIAV, IBDV, ARV, NDV, AIV, or other viruses, suggesting a high specificity for our method. Various titers of REV were artificially "spiked" into the FPV and MDV vaccines to simulate REV contamination in attenuated vaccines to validate this qRT-PCR method. Our findings indicated that this qRT-PCR method could detect REV contamination at a dose of 1 TCID50/1,000 feathers, which was 10,000-fold more sensitive than the regular RT-PCR detection (10(4) TCID50/1000 feathers). PMID:27122388

  12. Simultaneous detection of five notifiable viral diseases of cattle by single-tube multiplex real-time RT-PCR.

    PubMed

    Wernike, Kerstin; Hoffmann, Bernd; Beer, Martin

    2015-06-01

    Multiplexed real-time PCR (qPCR) assays enable the detection of several target genes in a single reaction, which is applicable for simultaneous testing for the most important viral diseases in samples obtained from ruminants with unspecific clinical symptoms. Here, reverse transcription qPCR (RT-qPCR) systems for the detection of bovine viral diarrhoea virus (BVDV) and bluetongue virus (BTV) were combined with an internal control system based on the beta-actin gene. Additionally, a background screening for three further major pathogens of cloven-hoofed animals reportable to the World Organisation for Animal Health, namely foot-and-mouth disease virus, epizootic haemorrhagic disease virus, and Rift Valley fever virus, was integrated using the identical fluorophore for the respective RT-qPCR assays. Every pathogen-specific assay had an analytical sensitivity of at least 100 genome copies per reaction within the multiplex approach, and a series of reference samples and clinical specimens obtained from cattle, but also from small ruminants, were detected reliably. The qPCR systems integrated in the background screening were even not influenced by the simultaneous amplification of very high BVDV and BTV genome copy numbers. The newly developed multiplex qPCR allows the specific and sensitive detection of five of the most important diseases of ruminants and could be used in the context of monitoring programs or for differential diagnostics. PMID:25746154

  13. Multiplex-PCR for simultaneous detection of 3 bacterial fish pathogens, Flavobacterium columnare, Edwardsiella ictaluri, and Aeromonas hydrophila.

    PubMed

    Panangala, Victor S; Shoemaker, Craig A; Van Santen, Vicky L; Dybvig, Kevin; Klesius, Phillip H

    2007-03-13

    A multiplex PCR (m-PCR) method was developed for simultaneous detection of 3 important fish pathogens in warm water aquaculture. The m-PCR to amplify target DNA fragments from Flavobacterium columnare (504 bp), Edwardsiella ictaluri (407 bp) and Aeromonas hydrophila (209 bp) was optimized by adjustment of reaction buffers and a touchdown protocol. The lower detection limit for each of the 3 bacteria was 20 pg of nucleic acid template from each bacteria per m-PCR reaction mixture. The sensitivity threshold for detection of the 3 bacteria in tissues ranged between 3.4 x 10(2) and 2.5 x 10(5) cells g(-1) of tissue (channel catfish Ictalurus punctatus Rafinesque). The diagnostic sensitivity and specificity of the m-PCR was evaluated with 10 representative isolates of each of the 3 bacteria and 11 other Gram-negative and 2 Gram-positive bacteria that are taxonomically related or ubiquitous in the aquatic environment. Except for a single species (A. salmonicida subsp. salmonicida), each set of primers specifically amplified the target DNA of the cognate species of bacteria. m-PCR was compared with bacteriological culture for identification of bacteria in experimentally infected fish. The m-PCR appears promising for the rapid, sensitive and simultaneous detection of Flavobacterium columnare, E. ictaluri and A. hydrophila in infected fish compared to the time-consuming traditional bacteriological culture techniques. PMID:17465305

  14. A duplex quantitative real-time PCR assay for the detection of Haplosporidium and Perkinsus species in shellfish.

    PubMed

    Xie, Zhixun; Xie, Liji; Fan, Qing; Pang, Yaoshan; Deng, Xianwen; Xie, Zhi Qin; Liu, Jiabo; Khan, Mazhar I

    2013-04-01

    A duplex quantitative real-time polymerase chain reaction (dq-PCR) assay was optimized to simultaneously detect Haplosporidium spp. and Perkinsus spp. of shellfish in one reaction. Two sets of specific oligonucleotide primers for Haplosporidium spp. and Perkinsus spp., along with two hydrolysis probes specific for each parasite group, were used in the assay. The dq-PCR results were detected and analyzed using the Light Cycler 2.0 software system. The dq-PCR identified and differentiated the two protozoan parasite groups. The sensitivity of the dq-PCR assay was 200 template copies for both Haplosporidium spp. and Perkinsus spp. No DNA product was amplified when known DNA from Marteilia refringens, Toxoplasma gondii, Bonamia ostreae, Escherichia coli, Cymndinium spp., Mykrocytos mackini, Vibrio parahaemolyticus, and shellfish tissue were used as templates. A total of 840 oyster samples from commercial cultivated shellfish farms from two coastal areas in China were randomly collected and tested by dq-PCR. The detection rate of Haplosporidium spp. was 8.6% in the Qindao, Shandong coastal area, whereas Perkinsus spp. was 8.3% coastal oysters cultivated from shellfish farms of Beihai, Guangxi. The dqPCR results suggested that Haplosporidium spp. was prevalent in oysters from Qindao, Shandong, while Perkinsus spp. was prevalent in oysters from the coastal areas of Beihai, Guangxi. This dq-PCR could be used as a diagnostic tool to detect Haplosporidium spp. and Perkinsus spp. in cultivated shellfish. PMID:23371501

  15. Group-Specific Multiplex PCR Detection Systems for the Identification of Flying Insect Prey

    PubMed Central

    Sint, Daniela; Niederklapfer, Bettina; Kaufmann, Ruediger; Traugott, Michael

    2014-01-01

    The applicability of species-specific primers to study feeding interactions is restricted to those ecosystems where the targeted prey species occur. Therefore, group-specific primer pairs, targeting higher taxonomic levels, are often desired to investigate interactions in a range of habitats that do not share the same species but the same groups of prey. Such primers are also valuable to study the diet of generalist predators when next generation sequencing approaches cannot be applied beneficially. Moreover, due to the large range of prey consumed by generalists, it is impossible to investigate the breadth of their diet with species-specific primers, even if multiplexing them. However, only few group-specific primers are available to date and important groups of prey such as flying insects have rarely been targeted. Our aim was to fill this gap and develop group-specific primers suitable to detect and identify the DNA of common taxa of flying insects. The primers were combined in two multiplex PCR systems, which allow a time- and cost-effective screening of samples for DNA of the dipteran subsection Calyptratae (including Anthomyiidae, Calliphoridae, Muscidae), other common dipteran families (Phoridae, Syrphidae, Bibionidae, Chironomidae, Sciaridae, Tipulidae), three orders of flying insects (Hymenoptera, Lepidoptera, Plecoptera) and coniferous aphids within the genus Cinara. The two PCR assays were highly specific and sensitive and their suitability to detect prey was confirmed by testing field-collected dietary samples from arthropods and vertebrates. The PCR assays presented here allow targeting prey at higher taxonomic levels such as family or order and therefore improve our ability to assess (trophic) interactions with flying insects in terrestrial and aquatic habitats. PMID:25525799

  16. Detection, quantification and vitality of Listeria monocytogenes in food as determined by quantitative PCR.

    PubMed

    Rantsiou, Kalliopi; Alessandria, Valentina; Urso, Rosalinda; Dolci, Paola; Cocolin, Luca

    2008-01-15

    In this paper we describe the development of a quantitative PCR (qPCR) technique to detect, quantify and determine the vitality of Listeria monocytogenes in foods. The method was based on the amplification of the intergenic region spacer (IGS) between the 16S and 23S rRNA genes. A panel of more than 100 strains of Listeria spp. and non-Listeria was used in order to verify the specificity of the primers and Taqman probe and amplification signals were obtained only when L. monocytogenes DNA and RNA were loaded in the qPCR mix. Standard curves were constructed in several food matrices (milk, meat, soft cheese, fermented sausage, cured ham and ready-to-eat salad). The quantification limit was of 10(3)-10(4) cfu/g or ml, while for the determination of vitality it was 10(4)-10(5) cfu/g or ml. After an overnight enrichment in BHI at 37 degrees C also 10 cfu/g or ml could be detected in all the matrices used in this study. When