The introduction of commercially available quantitative HIV-1 RNA detection methods at the end of the last century has had a significant impact on the management of patients requiring treatment. Similarly for hepatitis C virus (HCV), clinical decision-making with respect to initiation and prolonging therapy is largely based on data from viral load assays. The methods developed in the early 1990s and further improved since then still have significant drawbacks. For example, they are labor intensive, have a small dynamic range and are contamination sensitive. The development of real-time detection techniques for reverse transcription PCR has in part solved these problems. In the present review the advantages and disadvantages of the recently marketed Abbott Realtime HCV and HIV-1 viral load assays relative to their competitors will be discussed.
Tang, Ning; Frank, Andrea; Pahalawatta, Vihanga; Lampinen, John; Coblenz-Korte, Anke; Dunn, Chad; Li, Cheng; Cloherty, Gavin; Abravaya, Klara; Leckie, Gregor
Nucleic acid amplification test (NAAT)-based assays provide fast and sensitive results compared to conventional TB tests. The performance of the new Abbott Molecular MTB assay for the qualitative detection of MTB complex using the automated m2000™ system or manual sample preparation is summarized in this paper. The assay detects eight MTB complex subspecies. The observed limit of detection (LOD) when used to test an MTB H37Rv panel was 2.45 colony forming units (cfu)/mL, while the claimed assay LOD with this MTB strain is 17 cfu/mL. No cross reactivity, or carryover were observed in the study. The clinical sensitivity of the assay was 93% overall; 99% in smear positive, culture positive specimens, and 81% in smear negative, culture positive samples. The clinical specificity was 97%. The inhibition rate in the study was 0.34%. The data suggest that Abbott RealTime MTB is a reliable, robust and sensitive assay for the molecular detection of MTB. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.
Tang, Ning; Pahalawatta, Vihanga; Frank, Andrea; Bagley, Zowie; Viana, Raquel; Lampinen, John; Leckie, Gregor; Huang, Shihai; Abravaya, Klara; Wallis, Carole L
HIV RNA suppression is a key indicator for monitoring success of antiretroviral therapy. From a logistical perspective, viral load (VL) testing using Dried Blood Spots (DBS) is a promising alternative to plasma based VL testing in resource-limited settings. To evaluate the analytical and clinical performance of the Abbott RealTime HIV-1 assay using a fully automated one-spot DBS sample protocol. Limit of detection (LOD), linearity, lower limit of quantitation (LLQ), upper limit of quantitation (ULQ), and precision were determined using serial dilutions of HIV-1 Virology Quality Assurance stock (VQA Rush University), or HIV-1-containing armored RNA, made in venous blood. To evaluate correlation, bias, and agreement, 497 HIV-1 positive adult clinical samples were collected from Ivory Coast, Uganda and South Africa. For each HIV-1 participant, DBS-fingerprick, DBS-venous and plasma sample results were compared. Correlation and bias values were obtained. The sensitivity and specificity were analyzed at a threshold of 1000 HIV-1 copies/mL generated using the standard plasma protocol. The Abbott HIV-1 DBS protocol had an LOD of 839 copies/mL, a linear range from 500 to 1×10(7) copies/mL, an LLQ of 839 copies/mL, a ULQ of 1×10(7) copies/mL, and an inter-assay SD of ≤0.30 log copies/mL for all tested levels within this range. With clinical samples, the correlation coefficient (r value) was 0.896 between DBS-fingerprick and plasma and 0.901 between DBS-venous and plasma, and the bias was -0.07 log copies/mL between DBS-fingerprick and plasma and -0.02 log copies/mL between DBS-venous and plasma. The sensitivity of DBS-fingerprick and DBS-venous was 93%, while the specificity of both DBS methods was 95%. The results demonstrated that the Abbott RealTime HIV-1 assay with DBS sample protocol is highly sensitive, specific and precise across a wide dynamic range and correlates well with plasma values. The Abbott RealTime HIV-1 assay with DBS sample protocol provides an
Schnepf, Nathalie; Scieux, Catherine; Resche-Riggon, Matthieu; Feghoul, Linda; Xhaard, Alienor; Gallien, Sébastien; Molina, Jean-Michel; Socié, Gérard; Viglietti, Denis; Simon, François; Mazeron, Marie-Christine; Legoff, Jérôme
Fully standardized reproducible and sensitive quantification assays for cytomegalovirus (CMV) are needed to better define thresholds for antiviral therapy initiation and interruption. We evaluated the newly released Abbott RealTime CMV assay for CMV quantification in whole blood (WB) that includes automated extraction and amplification (m2000 RealTime system). Sensitivity, accuracy, linearity, and intra- and interassay variability were validated in a WB matrix using Quality Control for Molecular Diagnostics (QCMD) panels and the WHO international standard (IS). The intra- and interassay coefficients of variation were 1.37% and 2.09% at 5 log10 copies/ml and 2.41% and 3.80% at 3 log10 copies/ml, respectively. According to expected values for the QCMD and Abbott RealTime CMV methods, the lower limits of quantification were 104 and <50 copies/ml, respectively. The conversion factor between international units and copies (2.18), determined from serial dilutions of the WHO IS in WB, was significantly different from the factor provided by the manufacturer (1.56) (P = 0.001). Results from 302 clinical samples were compared with those from the Qiagen artus CMV assay on the same m2000 RealTime system. The two assays provided highly concordant results (concordance correlation coefficient, 0.92), but the Abbott RealTime CMV assay detected and quantified, respectively, 20.6% and 47.8% more samples than the Qiagen/artus CMV assay. The sensitivity and reproducibility of the results, along with the automation, fulfilled the quality requirements for implementation of the Abbott RealTime CMV assay in clinical settings. Our results highlight the need for careful validation of conversion factors provided by the manufacturers for the WHO IS in WB to allow future comparison of results obtained with different assays.
Schnepf, Nathalie; Scieux, Catherine; Resche-Riggon, Matthieu; Feghoul, Linda; Xhaard, Alienor; Gallien, Sébastien; Molina, Jean-Michel; Socié, Gérard; Viglietti, Denis; Simon, François; Mazeron, Marie-Christine
Fully standardized reproducible and sensitive quantification assays for cytomegalovirus (CMV) are needed to better define thresholds for antiviral therapy initiation and interruption. We evaluated the newly released Abbott RealTime CMV assay for CMV quantification in whole blood (WB) that includes automated extraction and amplification (m2000 RealTime system). Sensitivity, accuracy, linearity, and intra- and interassay variability were validated in a WB matrix using Quality Control for Molecular Diagnostics (QCMD) panels and the WHO international standard (IS). The intra- and interassay coefficients of variation were 1.37% and 2.09% at 5 log10 copies/ml and 2.41% and 3.80% at 3 log10 copies/ml, respectively. According to expected values for the QCMD and Abbott RealTime CMV methods, the lower limits of quantification were 104 and <50 copies/ml, respectively. The conversion factor between international units and copies (2.18), determined from serial dilutions of the WHO IS in WB, was significantly different from the factor provided by the manufacturer (1.56) (P = 0.001). Results from 302 clinical samples were compared with those from the Qiagen artus CMV assay on the same m2000 RealTime system. The two assays provided highly concordant results (concordance correlation coefficient, 0.92), but the Abbott RealTime CMV assay detected and quantified, respectively, 20.6% and 47.8% more samples than the Qiagen/artus CMV assay. The sensitivity and reproducibility of the results, along with the automation, fulfilled the quality requirements for implementation of the Abbott RealTime CMV assay in clinical settings. Our results highlight the need for careful validation of conversion factors provided by the manufacturers for the WHO IS in WB to allow future comparison of results obtained with different assays. PMID:23616450
Benedet, Marlin; Adachi, Dena; Wong, Anita; Wong, Sallene; Pabbaraju, Kanti; Tellier, Raymond; Tang, Julian W
Hepatitis C (HCV) genotyping is important for treatment planning. The Abbott m2000 RealTime HCV Genotype II assay is a PCR-based assay targeting specific regions of the 5'NCR gene for genotypes 1-6, and the NS5b gene for subgenotypes 1a/1b. However, not all genotypes can be resolved, with results being reported as: 'indeterminate', 'mixed', 'genotype X reactivity with Y', or just the major genotype 1 alone. To assess the supplementary testing required for these unresolved HCV genotypes, these samples were tested further using an in-house core/E1 sequencing assay. The resulting genotypes/subgenotypes were assigned using phylogenetic analysis with reference HCV genotype sequences. Additional testing was conducted using the INNO-LiPA HCV II assay for truly mixed genotypes. Out of 1052 samples tested, 89 (8.5%) underwent further sequencing to determine the HCV genotype: 16 that were 'indeterminate' on the m2000, were mostly genotype 2s and 3s by sequencing; 12 that were 'mixed', were mostly one of the genotypes reported in the mixture; 7 that were 'X reactivity with Y', were usually genotype X; 54 that gave just a major genotype 1 result were mostly 1a, with some 6 and 1b, and a few 1c. For three truly mixed genotypes, additional testing using the VERSANT(®) HCV Genotype Assay (LiPA) 2.0, showed two mixed 1 and 3, and one indistinguishable 6c-6l genotypes. The Abbott m2000 RealTime HCV Genotype II assay can resolve most (∼90%) HCV genotypes. However in 9-10% of cases, to fully resolve the genotype, additional testing is required. Copyright © 2014 Elsevier B.V. All rights reserved.
Schneider, George J; Kuper, Kevin G; Abravaya, Klara; Mullen, Carolyn R; Schmidt, Marion; Bunse-Grassmann, Astrid; Sprenger-Haussels, Markus
Automated sample preparation systems must meet the demands of routine diagnostics laboratories with regard to performance characteristics and compatibility with downstream assays. In this study, the performance of QIAGEN EZ1 DSP Virus Kit on the BioRobot EZ1 DSP was evaluated in combination with the Abbott RealTime HIV-1, HCV, and HBV assays, followed by thermalcycling and detection on the Abbott m2000rt platform. The following performance characteristics were evaluated: linear range and precision, sensitivity, cross-contamination, effects of interfering substances and correlation. Linearity was observed within the tested ranges (for HIV-1: 2.0-6.0 log copies/ml, HCV: 1.3-6.9 log IU/ml, HBV: 1.6-7.6 log copies/ml). Excellent precision was obtained (inter-assay standard deviation for HIV-1: 0.06-0.17 log copies/ml (>2.17 log copies/ml), HCV: 0.05-0.11 log IU/ml (>2.09 log IU/ml), HBV: 0.03-0.07 log copies/ml (>2.55 log copies/ml)), with good sensitivity (95% hit rates for HIV-1: 50 copies/ml, HCV: 12.5 IU/ml, HBV: 10 IU/ml). No cross-contamination was observed, as well as no negative impact of elevated levels of various interfering substances. In addition, HCV and HBV viral load measurements after BioRobot EZ1 DSP extraction correlated well with those obtained after Abbott m2000sp extraction. This evaluation demonstrates that the QIAGEN EZ1 DSP Virus Kit provides an attractive solution for fully automated, low throughput sample preparation for use with the Abbott RealTime HIV-1, HCV, and HBV assays.
Analytical and clinical performance characteristics of the Abbott RealTime MTB RIF/INH Resistance, an assay for the detection of rifampicin and isoniazid resistant Mycobacterium tuberculosis in pulmonary specimens.
Kostera, Joshua; Leckie, Gregor; Tang, Ning; Lampinen, John; Szostak, Magdalena; Abravaya, Klara; Wang, Hong
Clinical management of drug-resistant tuberculosis patients continues to present significant challenges to global health. To tackle these challenges, the Abbott RealTime MTB RIF/INH Resistance assay was developed to accelerate the diagnosis of rifampicin and/or isoniazid resistant tuberculosis to within a day. This article summarizes the performance of the Abbott RealTime MTB RIF/INH Resistance assay; including reliability, analytical sensitivity, and clinical sensitivity/specificity as compared to Cepheid GeneXpert MTB/RIF version 1.0 and Hain MTBDRplus version 2.0. The limit of detection (LOD) of the Abbott RealTime MTB RIF/INH Resistance assay was determined to be 32 colony forming units/milliliter (cfu/mL) using the Mycobacterium tuberculosis (MTB) strain H37Rv cell line. For rifampicin resistance detection, the Abbott RealTime MTB RIF/INH Resistance assay demonstrated statistically equivalent clinical sensitivity and specificity as compared to Cepheid GeneXpert MTB/RIF. For isoniazid resistance detection, the assay demonstrated statistically equivalent clinical sensitivity and specificity as compared to Hain MTBDRplus. The performance data presented herein demonstrate that the Abbott RealTime MTB RIF/INH Resistance assay is a sensitive, robust, and reliable test for realtime simultaneous detection of first line anti-tuberculosis antibiotics rifampicin and isoniazid in patient specimens.
Tam, Kingsley King-Gee; Leung, Kenneth Siu-Sing; To, Sabrina Wai-Chi; Siu, Gilman Kit-Hang; Lau, Terrence Chi-Kong; Shek, Victor Chi-Man; Tse, Cindy Wing-Sze; Wong, Samson Sai-Yin; Ho, Pak-Leung; Yam, Wing-Cheong
Abbott RealTime MTB (Abbott-RT) in conjunction with Abbott RealTime MTB RIF/INH Resistance (Abbott-RIF/INH) is a new, high-throughput automated nucleic acid amplification platform (Abbott-MDR) for detection of Mycobacterium tuberculosis complex (MTBC) and the genotypic markers for rifampicin (RIF) and isoniazid (INH) resistance directly from respiratory specimens. This prospective study evaluated the diagnostic performance of this new platform for MTBC and multidrug-resistant tuberculosis (MDR-TB) using 610 sputum specimens in a tuberculosis high-burden setting. Using conventional culture results and clinical background as reference standards, Abbott-RT exhibited an overall sensitivity and specificity of 95.2% and 99.8%, respectively. Genotypic RIF/INH resistance of 178 "MTB detected" specimens was subsequently analyzed by Abbott-RIF/INH. Compared to phenotypic drug susceptibility test results, Abbott-RIF/INH detected resistance genotypic markers in 84.6% MDR-TB, 80% mono-RIF-resistant and 66.7% mono-INH-resistant specimens. Two of the RIF-resistant specimens carried a novel single, nonsense mutation at rpoB Q513 and in silico simulation demonstrated that the truncated RpoB protein failed to bind with other subunits for transcription. Overall, Abbott-MDR platform provided high throughput and reliable diagnosis of MDR-TB within a TB high-burden region. Copyright © 2017 Elsevier Inc. All rights reserved.
Vinuesa, Víctor; Solano, Carlos; Giménez, Estela; Navarro, David
The ability of the artus Epstein-Barr virus (EBV) PCR kit and the Abbott RealTime EBV PCR assay to detect and quantify plasma EBV DNAemia was compared. The agreement between these assays was 95.8%. The EBV DNA loads measured by the two assays significantly correlated (P=< 0.0001).
Park, Seungman; Kang, Youjin; Kim, Dong Geun; Kim, Eui-Chong; Park, Sung Sup; Seong, Moon-Woo
The detection of high-risk (HR) HPV in cervical cancer screening is important for early diagnosis of cervical cancer or pre-cancerous lesions. We evaluated the analytical and clinical performances of 3 HR HPV assays in Gynecology patients. A total of 991 specimens were included in this study: 787 specimens for use with a Hybrid Capture 2 (HC2) and 204 specimens for a HPV DNA microarray (DNA Chip). All specimens were tested using an Abbott RealTime High Risk HPV assay (Real-time HR), PGMY PCR, and sequence analysis. Clinical sensitivities for severe abnormal cytology (severe than high-grade squamous intraepithelial lesion) were 81.8% for Real-time HR, 77.3% for HC2, and 66.7% for DNA Chip, and clinical sensitivities for severe abnormal histology (cervical intraepithelial neoplasia grade 2+) were 91.7% for HC2, 87.5% for Real-time HR, and 73.3% for DNA Chip. As compared to results of the sequence analysis, HC2, Real-time HR, and DNA Chip showed concordance rates of 94.3% (115/122), 90.0% (117/130), and 61.5% (16/26), respectively. The HC2 assay and Real-time HR assay showed comparable results to each other in both clinical and analytical performances, while the DNA Chip assay showed poor clinical and analytical performances. The Real-time HR assay can be a good alternative option for HR HPV testing with advantages of allowing full automation and simultaneous genotyping of HR types 16 and 18.
Impact of the New Abbott mPLUS Feature on Clinical Laboratory Efficiencies of Abbott RealTime Assays for Detection of HIV-1, Hepatitis C Virus, Hepatitis B Virus, Chlamydia trachomatis, and Neisseria gonorrhoeae
Jones, Sara; Wiesneth, Russ; Barry, Cathy; Webb, Erika; Belova, Larissa; Dolan, Peggy; Ho, Shiaolan; Abravaya, Klara; Cloherty, Gavin
Diagnostic laboratories are under increasing pressure to improve and expand their services. Greater flexibility in sample processing is a critical factor that can improve the time to results while reducing reagent waste, making laboratories more efficient and cost-effective. The introduction of the Abbott mPLUS feature, with the capacity for extended use of amplification reagents, significantly increases the flexibility of the m2000 platform and enables laboratories to customize their workflows based on sample arrival patterns. The flexibility in sample batch size offered by mPLUS enables significant reductions in processing times. For hepatitis B virus tests, a reduction in sample turnaround times of up to 30% (105 min) was observed for batches of 12 samples compared with those for batches of 24 samples; for Chlamydia trachomatis/Neisseria gonorrhoeae tests, the ability to run batches of 24 samples reduced the turnaround time by 83% (54 min) compared with that for batches of 48 samples. Excellent correlations between mPLUS and m2000 standard condition results were observed for all RealTime viral load assays evaluated in this study, with correlation r values of 0.998 for all assays tested. For the qualitative RealTime C. trachomatis/N. gonorrhoeae assay, the overall agreements between the two conditions tested were >98% for C. trachomatis and 100% for N. gonorrhoeae. Comparable precision results were observed for the two conditions tested for all RealTime assays. The enhanced mPLUS capability provides clinical laboratories with increased efficiencies to meet increasingly stringent turnaround time requirements without increased costs associated with discarding partially used amplification reagents. PMID:24088850
Vermehren, Johannes; Yu, Ming-Lung; Monto, Alexander; Yao, Joseph D; Anderson, Christopher; Bertuzis, Rasa; Schneider, George; Sarrazin, Christoph
Hepatitis C virus (HCV) RNA monitoring during antiviral therapy is essential for early prediction of treatment success and failure to peginterferon alfa/ribavirin (PEG-IFN/RBV) therapy. In this multi-center study we assessed the clinical utility of the Abbott RealTime HCV assay for monitoring patients undergoing antiviral therapy for chronic infection with HCV genotypes (GT) 1-3. We analyzed serum from 361 patients with chronic hepatitis C who had been treated with PEG-IFN/RBV. The predictive value of rapid virologic response (RVR), partial (≥2log(10) decline) and complete (HCV-RNA undetectable) early virologic response (pEVR/cEVR) based on RealTime HCV for achieving sustained virologic response was evaluated. In addition, the utility of RealTime HCV to tailor treatment duration according to individual virologic responses was studied in a subset of 136 GT 1 patients and compared to the reference tests, Versant HCV Quantitative 3.0 (bDNA) and Qualitative (TMA) assay. At week 4 of therapy, patients with RVR had a 100% and 93.5% probability to achieve an SVR in GT 1 and GT 2/3 patients, respectively. At week 12, patients who did not achieve a pEVR had a 97.2% and 100% probability of not achieving an SVR. In addition, assignment of GT 1 patients to abbreviated or extended treatment durations based on low baseline HCV-RNA (<800,000IU/mL) and RVR or pEVR was highly concordant between RealTime HCV and bDNA/TMA assays (97.8% and 91.9%, respectively). The RealTime HCV assay is suitable for monitoring virologic response to PEG-INF/RBV therapy and tailoring treatment duration accordingly. Copyright © 2011 Elsevier B.V. All rights reserved.
Worlock, A; Blair, D; Hunsicker, M; Le-Nguyen, T; Motta, C; Nguyen, C; Papachristou, E; Pham, J; Williams, A; Vi, M; Vinluan, B; Hatzakis, A
The Aptima HCV Quant Dx assay (Aptima assay) is a fully automated quantitative assay on the Panther® system. This assay is intended for confirmation of diagnosis and monitoring of HCV RNA in plasma and serum specimens. The purpose of the testing described in this paper was to evaluate the performance of the Aptima assay. The analytical sensitivity, analytical specificity, precision, and linearity of the Aptima assay were assessed. The performance of the Aptima assay was compared to two commercially available HCV assays; the Abbott RealTime HCV assay (Abbott assay, Abbott Labs Illinois, USA) and the Roche COBAS Ampliprep/COBAS Taqman HCV Quantitative Test v2.0 (Roche Assay, Roche Molecular Systems, Pleasanton CA, USA). The 95% Lower Limit of Detection (LoD) of the assay was determined from dilutions of the 2nd HCV WHO International Standard (NIBSC 96/798 genotype 1) and HCV positive clinical specimens in HCV negative human plasma and serum. Probit analysis was performed to generate the 95% predicted detection limits. The Lower Limit of Quantitation (LLoQ) was established for each genotype by diluting clinical specimens and the 2nd HCV WHO International Standard (NIBSC 96/798 genotype 1) in HCV negative human plasma and serum. Specificity was determined using 200 fresh and 536 frozen HCV RNA negative clinical specimens including 370 plasma specimens and 366 serum specimens. Linearity for genotypes 1 to 6 was established by diluting armored RNA or HCV positive clinical specimens in HCV negative serum or plasma from 8.08 log IU/mL to below 1 log IU/mL. Precision was tested using a 10 member panel made by diluting HCV positive clinical specimens or spiking armored RNA into HCV negative plasma and serum. A method comparison was conducted against the Abbott assay using 1058 clinical specimens and against the Roche assay using 608 clinical specimens from HCV infected patients. In addition, agreement between the Roche assay and the Aptima assay using specimens with low
Guichet, Emilande; Aghokeng, Avelin; Eymard-Duvernay, Sabrina; Vidal, Nicole; Ayouba, Ahidjo; Mpoudi Ngole, Eitel; Delaporte, Eric; Ciaffi, Laura; Peeters, Martine
With the increasing demand of HIV viral load (VL) tests in resource-limited countries (RLCs) there is a need for assays at affordable cost and able to quantify all known HIV-1 variants. VLs obtained with a recently developed open and polyvalent universal HIV-1/SIVcpz/SIVgor RT-qPCR were compared to Abbott RealTime HIV-1 assay in Cameroon. On 474 plasma samples, characterized by a wide range of VLs and a broad HIV-1 group M genetic diversity, 97.5% concordance was observed when using the lower detection limit of each assay. When using the threshold of 3.00 log10 copies/mL, according to WHO guidelines to define virological failure (VF) in RLCs, the concordance was 94.7%, 360/474 versus 339/474 patients were identified with VF with the new assay and Abbott RealTime HIV-1, respectively. Higher VLs were measured with the new assay, +0.47 log10 copies/mL (95% CI; 0.42-0.52) as shown with Bland-Altman analysis. Eleven samples from patients on VF with drug resistance were not detected by Abbott RealTime HIV-1 versus two only with the new assay. Overall, our study showed that the new assay can be easily implemented in a laboratory in RLCs with VL experience and showed good performance on a wide diversity of HIV-1 group M variants.
Yang, Ruifeng; Cong, Xu; Du, Shaocai; Fei, Ran; Rao, Huiying; Wei, Lai
The Versant HCV genotype 2.0 assay (line probe assay [LiPA] 2.0), based on reverse hybridization, and the Abbott Realtime HCV genotype II assay (Realtime II), based on genotype-specific real-time PCR, have been widely used to analyze hepatitis C virus (HCV) genotypes. However, their performances for detecting HCV genotype 6 infections have not been well studied. Here, we analyzed genotype 6 in 63 samples from the China HCV Genotyping Study that were originally identified as genotype 6 using the LiPA 2.0. The genotyping results were confirmed by nonstructural 5B (NS5B) or core sequence phylogenetic analysis. A total of 57 samples were confirmed to be genotype 6 (51 genotype 6a, 5 genotype 6n, and 1 genotype 6e). Four samples identified as a mixture of genotypes 6 and 4 by the LiPA 2.0 were confirmed to be genotype 3b. The remaining two samples classified as genotype 6 by the LiPA 2.0 were confirmed to be genotype 1b, which were intergenotypic recombinants and excluded from further comparison. In 57 genotype 6 samples detected using the Realtime II version 2.00 assay, 47 genotype 6a samples were identified as genotype 6, one 6e sample was misclassified as genotype 1, and four 6a and five 6n samples yielded indeterminate results. Nine nucleotide profiles in the 5' untranslated region affected the performances of both assays. Therefore, our analysis shows that both assays have limitations in identifying HCV genotype 6. The LiPA 2.0 cannot distinguish some 3b samples from genotype 6 samples. The Realtime II assay fails to identify some 6a and all non-6a subtypes, and it misclassifies genotype 6e as genotype 1. Copyright © 2014, American Society for Microbiology. All Rights Reserved.
Yang, Ruifeng; Cong, Xu; Du, Shaocai; Fei, Ran; Rao, Huiying
The Versant HCV genotype 2.0 assay (line probe assay [LiPA] 2.0), based on reverse hybridization, and the Abbott Realtime HCV genotype II assay (Realtime II), based on genotype-specific real-time PCR, have been widely used to analyze hepatitis C virus (HCV) genotypes. However, their performances for detecting HCV genotype 6 infections have not been well studied. Here, we analyzed genotype 6 in 63 samples from the China HCV Genotyping Study that were originally identified as genotype 6 using the LiPA 2.0. The genotyping results were confirmed by nonstructural 5B (NS5B) or core sequence phylogenetic analysis. A total of 57 samples were confirmed to be genotype 6 (51 genotype 6a, 5 genotype 6n, and 1 genotype 6e). Four samples identified as a mixture of genotypes 6 and 4 by the LiPA 2.0 were confirmed to be genotype 3b. The remaining two samples classified as genotype 6 by the LiPA 2.0 were confirmed to be genotype 1b, which were intergenotypic recombinants and excluded from further comparison. In 57 genotype 6 samples detected using the Realtime II version 2.00 assay, 47 genotype 6a samples were identified as genotype 6, one 6e sample was misclassified as genotype 1, and four 6a and five 6n samples yielded indeterminate results. Nine nucleotide profiles in the 5′ untranslated region affected the performances of both assays. Therefore, our analysis shows that both assays have limitations in identifying HCV genotype 6. The LiPA 2.0 cannot distinguish some 3b samples from genotype 6 samples. The Realtime II assay fails to identify some 6a and all non-6a subtypes, and it misclassifies genotype 6e as genotype 1. PMID:25100817
Rutstein, Sarah E.; Kamwendo, Deborah; Lugali, Lebah; Thengolose, Isaac; Tegha, Gerald; Fiscus, Susan A.; Nelson, Julie A. E.; Hosseinipour, Mina C.; Sarr, Abdoulaye; Gupta, Sundeep; Chimbwandira, Frank; Mwenda, Reuben; Mataya, Ronald
Background Viral suppression is a key indicator of antiretroviral therapy (ART) response among HIV-infected patients. Dried blood spots (DBS) are an appealing alternative to conventional plasma-based virologic testing, improving access to monitoring in resource-limited settings. However, validity of DBS obtained from fingerstick in field settings remains unknown. Objectives Investigate feasibility and accuracy of DBS vs plasma collected by healthcare workers in real-world settings of remote hospitals in Malawi. Compare venous DBS to fingerstick DBS for identifying treatment failure. Study design We recruited patients from ART clinics at two district hospitals in Malawi, collecting plasma, venous DBS (vDBS), and fingerstick DBS (fsDBS) cards for the first 149 patients, and vDBS and fsDBS only for the subsequent 398 patients. Specimens were tested using Abbott RealTime HIV-1 Assay (lower detection limit 40 copies/ml (plasma) and 550 copies/ml (DBS)). Results 21/149 (14.1%) had detectable viremia (>1.6 log copies/ml), 13 of which were detectable for plasma, vDBS, and fsDBS. Linear regression demonstrated high correlation for plasma vs. DBS (vDBS: β=1.19, R2 0.93 (p<0.0001); fsDBS β=1.20, R2 0.90 (p<0.0001)) and vDBS vs. fsDBS (β=0.88, R2 0.73, (p<0.0001)). Mean difference between plasma and vDBS was 0.51 log copies/ml [SD: 0.33] and plasma and fsDBS 0.46 log copies/ml [SD: 0.30]. At 5000 copies/ml, sensitivity was 100%, and specificity was 98.6% and 97.8% for vDBS and fsDBS, respectively, compared to plasma. Conclusions DBS from venipuncture and fingerstick perform well at the failure threshold of 5000 copies/ml. Fingerstick specimen source may improve access to virologic treatment monitoring in resource-limited settings given task-shifting in high-volume, low-resource facilities. PMID:24906641
Hofmann-Thiel, Sabine; Molodtsov, Nikolay; Antonenka, Uladzimir; Hoffmann, Harald
The Abbott RealTime MTB (RT MTB) assay is a new automated nucleic acid amplification test for the detection of Mycobacterium tuberculosis complex (MTBC) in clinical specimens. In combination with the RealTime MTB INH/RIF (RT MTB INH/RIF) resistance assay, which can be applied to RT MTB-positive specimens as an add-on assay, the tests also indicate the genetic markers of resistance to isoniazid (INH) and rifampin (RIF). We aimed to evaluate the diagnostic sensitivity and specificity of RT MTB using different types of respiratory and extrapulmonary specimens and to compare performance characteristics directly with those of the FluoroType MTB assay. The resistance results obtained by RT MTB INH/RIF were compared to those from the GenoType MTBDRplus and from phenotypic drug susceptibility testing. A total of 715 clinical specimens were analyzed. Compared to culture, the overall sensitivity of RT MTB was 92.1%; the sensitivity rates for smear-positive and smear-negative samples were 100% and 76.2%, respectively. The sensitivities of smear-negative specimens were almost identical for respiratory (76.3%) and extrapulmonary (76%) specimens. Specificity rates were 100% and 95.8% for culture-negative specimens and those that grew nontuberculous mycobacteria, respectively. RT MTB INH/RIF was applied to 233 RT MTB-positive samples and identified resistance markers in 7.7% of samples. Agreement with phenotypic and genotypic drug susceptibility testing was 99.5%. In conclusion, RT MTB and RT MTB INH/RIF allow for the rapid and accurate diagnosis of tuberculosis (TB) in different types of specimens and reliably indicate resistance markers. The strengths of this system are the comparably high sensitivity with paucibacillary specimens, its ability to detect INH and RIF resistance, and its high-throughput capacities.
Park, Yongjung; Lee, Eunhee; Choi, Jonghyeon; Jeong, Seri; Kim, Hyon-Suk
Infection with high-risk (HR) human papillomavirus (HPV) genotypes is an important risk factor for cervical cancers. We evaluated the clinical performances of two new real-time PCR assays for detecting HR HPVs compared to that of the Hybrid Capture 2 test (HC2). A total of 356 cervical swab specimens, which had been examined for cervical cytology, were assayed by Abbott RealTime HR and Roche Cobas HPV as well as HC2. Sensitivities and specificities of these assays were determined based on the criteria that concordant results among the three assays were regarded as true-positive or -negative and that the results of genotyping and sequencing were considered true findings when the HPV assays presented discrepant results. The overall concordance rate among the results for the three assays was 82.6%, and RealTime HR and Cobas HPV assays agreed with HC2 in 86.1% and 89.9% of cases, respectively. The two real-time PCR assays agreed with each other for 89.6% of the samples, and the concordance rate between them was equal to or greater than 98.0% for detecting HPV type 16 or 18. HC2 demonstrated a sensitivity of 96.6% with a specificity of 89.1% for detecting HR HPVs, while RealTime HR presented a sensitivity of 78.3% with a specificity of 99.2%. The sensitivity and specificity of Cobas HPV for detecting HR HPVs were 91.7% and 97.0%. The new real-time PCR assays exhibited lower sensitivities for detecting HR HPVs than that of HC2. Nevertheless, the newly introduced assays have an advantage of simultaneously identifying HPV types 16 and 18 from clinical samples.
Rapid virological response assessment by Abbott RealTime hepatitis C virus assay for predicting sustained virological responses in patients with hepatitis C virus genotype 1 treated with pegylated-interferon and ribavirin.
Su, Pei-Yuan; Yen, Hsu-Heng; Hsu, Yu-Chun; Wu, Shun-Sheng; Kor, Chew-Teng; Su, Wei-Wen
The lower limits of virus detection of hepatitis C virus (HCV) RNA detection assays are continuously improving. We aimed to assess the utility of more precise definition of 4(th) week viral load [rapid virological response (RVR)] in predicting sustained virological response (SVR) in HCV genotype 1 patients treated with pegylated-interferon (PEG-IFN) and ribavirin. Clinical data of treatment-naïve HCV genotype 1 patients were retrospectively collected from 2009 to 2014. Patients were grouped according to 4(th) week viral load as follows: undetectable (n = 90) and detectable but not quantifiable (< 12 IU/mL, n = 27). All patients received PEG-IFNα-2a or -2b and ribavirin for 24 weeks. Serum HCV RNA levels were measured by Abbott RealTime (ART; Abbott Molecular, Abbott Park, IL, USA) HCV assay. SVR was 95.5% and 63% in the undetectable group and < 12 IU/mL group of 4(th) week viral load, respectively. The between-group difference in SVR was significant (p < 0.001). We determined 4(th) week viral load was independently associated with SVR (odds ratio = 19.28; p = 0.002) and a good predictor of SVR [area under the curve (AUC) = 0.775; p = 0.001]. ART HCV assays had a stronger SVR predictive value in HCV genotype 1 patients, indicating that only the undetectable group of 4(th) week viral load patients measured by ART HCV assay should be considered for shorter treatment time (24 weeks) with PEG-IFN and ribavirin.
Yeh, Ming-Lun; Huang, Chung-Feng; Huang, Ching-I; Liu, Shu-Fen; Yang, Hua-Ling; Hsieh, Ming-Yen; Huang, Jee-Fu; Dai, Chia-Yen; Chuang, Wan-Long; Yu, Ming-Lung
Selection of drug-resistant strains may lead to failure of HBV antiviral therapy. There is little information whether there is detection difference in drug resistant mutations between different viral load assays of HBV. This study is aimed to investigate whether there is drug-resistant strains related detection difference between Abbott RealTime HBV (RealTime) and CobasAmpliPrep/CobasTaqMan HBV assays 2.0 (TaqMan). One hundred and thirty-four CHB patients who received HBV anti-viral therapy were enrolled. HBV virological markers were tested 3 months apart regularly. Serum HBV DNA levels were determined using the TaqMan and RealTime. YMDD (rt180M and rt204V) mutation was checked in patients who experienced virologic breakthrough (VBT). The correlation of HBV DNA observed between the RealTime and TaqMan was good for all 571 samples (R2 = 0.797; P<0.001). However, the correlation in the 434 samples with HBV DNA level <3 log10 IU/ml was not as good as in all samples (R2 = 0.457). Overall, 21.5% of samples had a detection difference of ≥ 1 log10 IU/ml with 91.9% of these having HBV DNA level <3 log10 IU/ml. Twenty-four patients experienced VBT. Three of these patients had acquired the YMDD mutation and exhibited discordant viral load results between the two methods tested. In each case, persistent HBV DNA was detected by RealTime and undetectable with TaqMan. Of the patients who experienced a VBT and had acquired YMDD mutation, 4.7% had undetectable HBV DNA by TaqMan while all were detectable with RealTime. RealTime assay is more sensitive and is little impacted by the development of drug resistant mutation.
Abbott RealTime Hepatitis C Virus (HCV) and Roche Cobas AmpliPrep/Cobas TaqMan HCV Assays for Prediction of Sustained Virological Response to Pegylated Interferon and Ribavirin in Chronic Hepatitis C Patients ▿
Matsuura, Kentaro; Tanaka, Yasuhito; Hasegawa, Izumi; Ohno, Tomoyoshi; Tokuda, Hiroshi; Kurbanov, Fuat; Sugauchi, Fuminaka; Nojiri, Shunsuke; Joh, Takashi; Mizokami, Masashi
Two commercial real-time PCR assays are currently available for sensitive hepatitis C virus (HCV) RNA quantification: the Abbott RealTime HCV assay (ART) and Roche Cobas AmpliPrep/Cobas TaqMan HCV assay (CAP/CTM). We assessed whether the two real-time PCR assays were more effective than Roche Cobas Amplicor HCV Monitor test, v.2.0 (CAM) for prediction of the sustained virological response (SVR) to pegylated interferon (PEG-IFN) plus ribavirin (RBV) in chronic hepatitis C. Sixty patients chronically infected with HCV genotype 1b (37 males and 23 females, 53 ± 12 years of age) were treated with PEG-IFNα2b plus RBV for 48 weeks. Stored specimens at nine time points for each patient (at baseline, on treatment, and 24 weeks after treatment) were tested by the two real-time PCR assays and CAM. Twenty-six (43.3%) patients reached SVR. The positive predictive values (PPVs) for SVR of undetectable HCV RNA at week 12 by CAM, ART, and CAP/CTM were 74.3%, 88.0%, and 95.2%, respectively. An undetectable HCV RNA level by CAM, ART, and CAP/CTM correctly predicted SVR at week 4 in 100%, 100%, and 100% of patients, at weeks 5 to 8 in 91.7%, 100%, and 100% of patients, at weeks 9 to 12 in 55.6%, 75%, and 87.5% of patients, and at weeks 13 to 24 in 0%, 26.7%, and 40% of patients, respectively. Of 16 patients who relapsed after treatment, HCV RNA was detectable in 2 patients at the end of treatment by CAP/CTM but undetectable by ART and CAM. HCV RNA tests using ART and CAP/CTM are considered to be more effective at predicting SVR than CAM, and the PPV for SVR was slightly higher in CAP/CTM than in ART. PMID:19091819
Germer, Jeffrey J; Abraham, Priya; Mandrekar, Jayawant N; Yao, Joseph D C
The Abbott HBV RUO Sequencing assay (Abbott Molecular Inc., Des Plaines, IL), which combines automated sample processing, real-time PCR, and bidirectional DNA sequencing, was evaluated for detection of nucleos(t)ide analogue (NA) resistance-associated mutations located in the hepatitis B virus (HBV) polymerase (Pol) gene. Interpretive software from the assay manufacturer was modified to allow interrogation of the overlapping HBV surface (S) gene sequence for HBV genotype determination and detection of immune escape mutations. Analytical sensitivity (detection and sequencing) of the assay was determined to be 103.9 IU/ml (95% confidence interval [CI], 80.0 to 173.3) for HBV genotype A. Testing of commercially available HBV genotype panels consisting of 23 individual members yielded complete agreement between expected results and results obtained from the laboratory-developed HBV genotype library. Excellent specificity was observed among clinical specimens with serologic or molecular markers for various unrelated blood-borne viruses (n = 6) and sera obtained from healthy, HBV-negative blood donors (n = 20). Retrospectively selected clinical specimens tested by a commercial reference laboratory HBV sequencing assay (n = 54) or the Trugene HBV Genotyping kit (n = 7) and the Abbott HBV RUO Sequencing assay showed minor differences in detection and reporting of NA resistance-associated mutations in 7 of 61 (11.5%) specimens but complete agreement of genotype results. The Abbott HBV RUO Sequencing assay provided a convenient and efficient assay workflow suitable for routine clinical laboratory use, with the flexibility to be modified for customized detection of NA resistance-associated mutations, HBV genotype determination, and detection of immune escape mutations from a single contiguous HBV sequence.
Ability of two commercially available assays (Abbott RealTime HIV-1 and Roche Cobas AmpliPrep/Cobas TaqMan HIV-1 Version 2.0) to quantify low HIV-1 RNA Levels (<1,000 copies/milliliter): comparison with clinical samples and NIBSC working reagent for nucleic acid testing assays.
Amendola, Alessandra; Marsella, Patrizia; Bloisi, Maria; Forbici, Federica; Angeletti, Claudio; Capobianchi, Maria R
Concordance between molecular assays may be suboptimal at low HIV-1 viremia levels (<1,000 copies/ml); therefore, it may be difficult to define and compare virologic endpoints for successful and failed therapy. We compared two commercial assays (the Abbott RealTime HIV-1 and the Roche Cobas AmpliPrep/TaqMan HIV-1 version 2.0) for their ability to detect and quantify low viral loads. A comparison was performed using 167 residual clinical samples (with values ranging from "not detected" to 1,000 copies/ml, as measured by the Abbott assay) and the National Institute and Biological Standards and Control (NIBSC) HIV-1 RNA working reagent 1 for nucleic acid amplification techniques (NAT) assays (serially diluted to a range from 1 to 1,000 copies/ml). Quantitative results were compared using Lin's concordance correlation coefficient and a Bland-Altman plot. Concordance with the qualitative results was measured by Cohen's kappa statistic. With clinical samples, the degree of interassay concordance of the qualitative results at a 40-copies/ml HIV-1 RNA threshold was substantial (κ = 0.762); the correlation among the quantified samples was suboptimal (concordance correlation coefficient, 0.728; P < 0.0001); the mean difference of the values between the Roche and Abbott assays was 0.193 log10 copies/ml. Using the HIV-1 RNA working reagent 1 for NAT assays, the results provided by the Roche assay were, on average, 3 times higher than expected, while the Abbott assay showed high accuracy. The Roche assay was highly sensitive, being able to detect a level as low as 3.5 copies/ml HIV-1 RNA with 95% probability. The performance characteristics of each molecular assay should be taken into account when HIV-1 RNA threshold values for "virologic suppression," "virologic failure," "persistent low viral loads," etc., are defined and indicated in the support of clinical decisions. Copyright © 2014, American Society for Microbiology. All Rights Reserved.
Evaluation of performance across the dynamic range of the Abbott RealTime HIV-1 assay as compared to VERSANT HIV-1 RNA 3.0 and AMPLICOR HIV-1 MONITOR v1.5 using serial dilutions of 39 group M and O viruses.
Swanson, Priscilla; Huang, Shihai; Abravaya, Klara; de Mendoza, Carmen; Soriano, Vincent; Devare, Sushil G; Hackett, John
Performance of the Abbott m2000 instrument system and the Abbott RealTime HIV-1 assay was evaluated using a panel of 37 group M (subtypes A-D, F, G, CRF01_AE, CRF02_AG and unique recombinant forms) and 2 group O virus isolates. Testing was performed on 273 sample dilutions and compared to VERSANT HIV-1 RNA 3.0 (bDNA) and AMPLICOR HIV-1 MONITOR v1.5 (Monitor v1.5) test results. RealTime HIV-1, bDNA, and Monitor v1.5 tests quantified 87%, 78%, and 81% of samples, respectively. RealTime HIV-1 detected an additional 31 samples at < 40 copies/mL. For group M, RealTime HIV-1 dilution profiles and viral loads were highly correlated with bDNA and Monitor v1.5 values; 87% and 89% of values were within 0.5 log(10) copies/mL. In contrast, the group O viruses were not detected by Monitor v1.5 and were substantially underquantified by approximately 2 log(10) copies/mL in bDNA relative to the RealTime HIV-1 assay. Sequence analysis revealed that RealTime HIV-1 primer/probe binding sites are highly conserved and exhibit fewer nucleotide mismatches relative to Monitor v1.5. The automated m2000 system and RealTime HIV-1 assay offer the advantages of efficient sample processing and throughput with reduced "hands-on" time while providing improved sensitivity, expanded dynamic range and reliable quantification of genetically diverse HIV-1 strains.
Comparison of the clinical performances of the AdvanSure HPV Screening Real-Time PCR, the Abbott Real-Time High-Risk HPV Test, and the Hybrid Capture High-Risk HPV DNA Test for Cervical Cancer Screening.
Chung, Hae-Sun; Hahm, Chorong; Lee, Miae
The clinical performance of three human papillomavirus (HPV) DNA commercial assays for cervical cancer screening was evaluated; the AdvanSure HPV Screening Real-Time PCR (AdvanSure PCR; LG Life Sciences) that was developed recently for the detection of both high-risk and low-risk genotypes, the Abbott RealTime High-Risk HPV Test (Abbott PCR; Abbott Molecular) and the Hybrid Capture High-Risk HPV DNA test (HC2; Qiagen). The three different HPV DNA tests were compared using cytology samples obtained from 619 women who underwent routine cervical cancer screening. The gold-standard assay was histopathological confirmation of cervical intraepithelial neoplasia of grade 2 or worse. The clinical sensitivities of the AdvanSure PCR, the Abbott PCR and the HC2 for the detection of cervical intraepithelial neoplasia of grade 2 or worse were 95.5%, 95.5% and 100%, respectively, while the clinical specificities were 61.6%, 86.4% and 83.3%, respectively. There were no significant differences in the clinical sensitivities of the Abbott PCR and the AdvanSure PCR compared to the HC2. The clinical specificities of the Abbott PCR and the AdvanSure PCR for the detection of HPV types 16/18 were 97.8% and 98.5%, respectively. For cervical cancer screening, all three tests showed relatively good clinical sensitivities, but the AdvanSure PCR had lower clinical specificity than the Abbott PCR and the HC2. The AdvanSure PCR and the Abbott PCR assays have the advantage of being automated and the ability to distinguish between HPV types 16/18 and other HPV types. The two real-time PCR assays could be useful tools in HPV testing for cervical cancer screening.
Li, Z Y; Yan, C L; Yan, R; Feng, Z R
Monitoring of tacrolimus concentrations has played a crucial role to control the efficacy versus adverse effects of treatment, because of the drug's narrow therapeutic range, inter- and intraindividual variabilities, and drug interactions mediated by hepatic cytochrome P450. Therefore, the stability of methods to monitor tacrolimus is of major importance. This study evaluated the analytical performance of the Abbott Architect i2000 tacrolimus assay, which was recently released in China to monitor tacrolimus therapy. We compared the results using the Abbott Architect i2000 Tacrolimus assay with the traditional Abbott IMx method. We measured concentrations of tacrolimus in 100 samples from renal transplant patients by Architect i2000 and the commonly used Abbott IMx. We observed that the total %CV of the Architect i2000 assay was <10%, both at low and high concentration levels, with a functional sensitivity of <0.5 ng/mL. The Architect i2000 assay was linear over the reportable range with recoveries ranging from 90.6% to 106.7%. In addition, this assay was not affected by high concentrations of hemoglobin, lipids, or bilirubin in the samples. The 2 assays yielded similar results. The regression equation obtained from the Passing Bablok analysis was: y = 0.8788x - 0.3485 with a Spearman correlation coefficient (r) of 0.9922. The bias plot between the Architect i2000 and Abbott IMx assay yielded an average negative bias (-1.4 ng/mL). We concluded that, because of its high precision and sensitivity, low cross-reactivity, and acceptable accuracy, the Architect i2000 assay is a suitable alternative to monitor tacrolimus concentrations in renal transplant recipients. Copyright © 2010 Elsevier Inc. All rights reserved.
Azzazy, H M; Chou, P P; Tsushima, J H; Troxil, S; Gordon, M; Avers, R J; Chiappetta, E; Duh, S H; Christenson, R H
The authors evaluated the performance characteristics of the Abbott AxSYM Vancomycin II immunoassay in sera of patients with (n = 93 samples) and without (n = 327 patients) renal dysfunction. Correlation of vancomycin measurements with the Abbott AxSYM Vancomycin, Abbott TDx/TDxFLx, Syva enzyme-multiplied immunoassay technique (EMIT), DuPont automated chemistry analyzer (ACA), and high-performance liquid chromatography methods showed acceptable correlation as indicated by: slope values >0.95, r-values >0.97, y-intercepts <1.7 microg/ml, and S(y/x) ranging from 9% to 15% of the average vancomycin value. The AxSYM Vancomycin II assay showed acceptable correlation with AxSYM vancomycin, TDx/TDxFLx, and high-performance liquid chromatography methods in 93 samples from patients with renal dysfunction. This monoclonal antibody-based assay showed no apparent interference from the presence of human antimouse antibody (HAMA) or the microbiologically inactive vancomycin crystalline degradation product (CDP). The authors conclude that the AxSYM Vancomycin II assay showed satisfactory agreement with other methods tested in this study.
Hutchinson, Katrina; Healy, Martin; Crowley, Vivion; Louw, Michael; Rochev, Yury
Analytical and clinical verification of both old and new generations of the Abbott total 25-hydroxyvitamin D (25OHD) assays, and an examination of reference Intervals. Determination of between-run precision, and Deming comparison between patient sample results for 25OHD on the Abbott Architect, DiaSorin Liaison and AB SCIEX API 4000 (LC-MS/MS). Establishment of uncertainty of measurement for 25OHD Architect methods using old and new generations of the reagents, and estimation of reference interval in healthy Irish population. For between-run precision the manufacturer claims 2.8% coefficients of variation (CVs) of 2.8% and 4.6% for their high and low controls, respectively. Our instrument showed CVs between 4% and 6.2% for all levels of the controls on both generations of the Abbott reagents. The between-run uncertainties were 0.28 and 0.36, with expanded uncertainties 0.87 and 0.98 for the old and the new generations of reagent, respectively. The difference between all methods used for patients' samples was within total allowable error, and the instruments produced clinically equivalent results. The results covered the medical decision points of 30, 40, 50 and 125 nmol/L. The reference interval for total 25OHD in our healthy Irish subjects was lower than recommended levels (24-111 nmol/L). In a clinical laboratory Abbott 25OHD immunoassays are a useful, rapid and accurate method for measuring total 25OHD. The new generation of the assay was confirmed to be reliable, accurate, and a good indicator for 25OHD measurement. More study is needed to establish reference intervals that correctly represent the healthy population in Ireland.
A European multicientre study on the comparison of HBV viral loads between VERIS HBV assay and Roche COBAS(®) TAQMAN(®) HBV test, Abbott RealTime HBV assay, Siemens VERSANT HBV assay, and Qiagen artus HBV RG kit.
Braun, Patrick; Delgado, Rafael; Drago, Monica; Fanti, Diana; Fleury, Hervé; Izopet, Jacques; Lombardi, Alessandra; Marcos, MaAngeles; Sauné, Karine; O'Shea, Siobhan; Pérez-Rivilla, Alfredo; Ramble, John; Trimoulet, Pascale; Vila, Jordi; Whittaker, Duncan; Artus, Alain; Rhodes, Daniel
Hepatitis B viral load testing is essential to treatment and monitoring decisions in patients with chronic Hepatitis B. Beckman Coulter has developed the VERIS HBV Assay (Veris) for use on the fully automated DxN VERIS Molecular Diagnostics System.(1) OBJECTIVES: To evaluate the clinical performance of the Veris HBV Assay at multiple EU laboratories STUDY DESIGN: Method comparison was performed with a total of 344 plasma specimens from HBV infected patients tested with Veris and COBAS(®) TaqMan(®) HBV Test (Cobas), 207 specimens tested with Veris and RealTime HBV Assay (RealTime), 86 specimens tested with Veris and VERSANT(®) HBV Assay (Versant), and 74 specimens tested with Veris and artus(®) HBV RG PCR kit (artus). Bland-Altman analysis showed average bias of -0.46 log10 IU/mL between Veris and Cobas, -0.46 log10IU/mL between Veris and RealTime, -0.36 log10IU/mL between Veris and Versant, and -0.12 log10IU/mL between Veris and artus. Bias was consistent across the assay range. Patient monitoring results using Veris demonstrated similar viral load trends over time to Cobas, RealTime, and artus. The VERIS HBV Assay demonstrated comparable clinical performance, with varying degrees of negative bias, compared to other currently marketed assays for HBV DNA monitoring. This negative bias should be taken into consideration if switching monitoring methods to Veris. Copyright © 2017 Elsevier B.V. All rights reserved.
Flodin, Mats; Larsson, Anders
Glomerular filtration rate (GFR) is widely accepted as the best overall measure of kidney function. Cystatin C is a novel endogenous GFR marker that has been shown to be superior to creatinine for estimation of GFR in several studies. There is a need for cystatin C assays adapted to routine chemistry instrument to minimize turnaround times and allowing 24 h/day availability. We have evaluated a new cystatin C assay developed for Architect cSystem (Abbott Laboratories, Abbott Park, IL, USA). The cystatin C assay showed good agreement with the corresponding assay from Dade Behring (Deerfield, IL, USA). The assay has a very low total imprecision and a good linearity. The new cystatin C assay is an interesting alternative to current cystatin C assays. On an Architect cSystem the assay can be performed with the same turnaround times and availability as creatinine.
Boer, Klas; Deufel, Thomas; Schmidt, Dirk; Streck, Sibylle; Kiehntopf, Michael
Evaluation of the performance of the EMIT 2000 Tacrolimus assay on the Abbott Architect c8000 analyzer. Imprecision studies were performed and patient samples were assayed by EMIT assay and by LC-MS/MS. Limit of quantification was established at 2.8 microg/L. A positive bias of 17.5% between results measured on EMIT and on LC-MS/MS was detected. EMIT 2000 Tacrolimus assay is suitable for automated analyses of Tacrolimus on the Architect c8000.
De Monte, Anne; Cannavo, Isabelle; Caramella, Anne; Ollier, Laurence; Giordanengo, Valérie
Congenital cytomegalovirus (CMV) infection is the leading cause of sensoneurinal disability due to infectious congenital disease. The diagnosis of congenital CMV infection is based on the search of CMV in the urine within the first two weeks of life. Viral culture of urine is the gold standard. However, the PCR is highly sensitive and faster. It is becoming an alternative choice. The objective of this study is the validation of real-time PCR by Abbott RealTime CMV with m2000 for the detection of cytomegalovirus in urine. Repeatability, reproducibility, detection limit and inter-sample contamination were evaluated. Urine samples from patients (n=141) were collected and analyzed simultaneously in culture and PCR in order to assess the correlation of these two methods. The sensitivity and specificity of PCR were also calculated. The Abbott RealTime CMV PCR in urine is an automated and sensitive method (detection limit 200 UI/mL). Fidelity is very good (standard deviation of repeatability: 0.08 to 0.15 LogUI/mL and reproducibility 0.18 LogUI/mL). We can note a good correlation between culture and Abbott RealTime CMV PCR (kappa 96%). When considering rapid culture as reference, real-time PCR was highly sensitive (100%) and specific (98.2%). The real-time PCR by Abbott RealTime CMV with m2000 is optimal for CMV detection in urine.
Kapke, G E; Watson, G; Sheffler, S; Hunt, D; Frederick, C
Several assays for quantification of DNA have been developed and are currently used in research and clinical laboratories. However, comparison of assay results has been difficult owing to the use of different standards and units of measurements as well as differences between assays in dynamic range and quantification limits. Although a few studies have compared results generated by different assays, there has been no consensus on conversion factors and thorough analysis has been precluded by small sample size and limited dynamic range studied. In this study, we have compared the Chiron branched DNA (bDNA) and Abbott liquid hybridization assays for quantification of hepatitis B virus (HBV) DNA in clinical specimens and have derived conversion factors to facilitate comparison of assay results. Additivity and variance stabilizing (AVAS) regression, a form of non-linear regression analysis, was performed on assay results for specimens from HBV clinical trials. Our results show that there is a strong linear relationship (R2 = 0.96) between log Chiron and log Abbott assay results. Conversion factors derived from regression analyses were found to be non-constant and ranged from 6-40. Analysis of paired assay results below and above each assay's limit of quantification (LOQ) indicated that a significantly (P < 0.01) larger proportion of observations were below the Abbott assay LOQ but above the Chiron assay LOQ, indicating that the Chiron assay is significantly more sensitive than the Abbott assay. Testing of replicate specimens showed that the Chiron assay consistently yielded lower per cent coefficients of variance (% CVs) than the Abbott assay, indicating that the Chiron assay provides superior precision.
Espino, A. A.; Maceira, V. P.; Nattanmai, S. M.; Butt, S. A.; Wroblewski, D.; Hannett, G. E.; Musser, K. A.
Clostridium difficile-associated diarrhea is a well-recognized complication of antibiotic use. Historically, diagnosing C. difficile has been difficult, as antigen assays are insensitive and culture-based methods require several days to yield results. Nucleic acid amplification tests (NAATs) are quickly becoming the standard of care. We compared the performance of two automated investigational/research use only (IUO/RUO) NAATs for the detection of C. difficile toxin genes, the IMDx C. difficile for Abbott m2000 Assay (IMDx) and the BD Max Cdiff Assay (Max). A prospective analysis of 111 stool specimens received in the laboratory for C. difficile testing by the laboratory's test of record (TOR), the BD GeneOhm Cdiff Assay, and a retrospective analysis of 88 specimens previously determined to be positive for C. difficile were included in the study. One prospective specimen was excluded due to loss to follow-up discrepancy analysis. Of the remaining 198 specimens, 90 were positive by all three methods, 9 were positive by TOR and Max, and 3 were positive by TOR only. One negative specimen was initially inhibitory by Max. The remaining 95 specimens were negative by all methods. Toxigenic C. difficile culture was performed on the 12 discrepant samples. True C. difficile-positive status was defined as either positive by all three amplification assays or positive by toxigenic culture. Based on this definition, the sensitivity and specificity were 96.9% and 95% for Max and 92.8% and 100% for IMDx. In summary, both highly automated systems demonstrated excellent performance, and each has individual benefits, which will ensure that they will both have a niche in clinical laboratories. PMID:24554744
Stellrecht, K A; Espino, A A; Maceira, V P; Nattanmai, S M; Butt, S A; Wroblewski, D; Hannett, G E; Musser, K A
Clostridium difficile-associated diarrhea is a well-recognized complication of antibiotic use. Historically, diagnosing C. difficile has been difficult, as antigen assays are insensitive and culture-based methods require several days to yield results. Nucleic acid amplification tests (NAATs) are quickly becoming the standard of care. We compared the performance of two automated investigational/research use only (IUO/RUO) NAATs for the detection of C. difficile toxin genes, the IMDx C. difficile for Abbott m2000 Assay (IMDx) and the BD Max Cdiff Assay (Max). A prospective analysis of 111 stool specimens received in the laboratory for C. difficile testing by the laboratory's test of record (TOR), the BD GeneOhm Cdiff Assay, and a retrospective analysis of 88 specimens previously determined to be positive for C. difficile were included in the study. One prospective specimen was excluded due to loss to follow-up discrepancy analysis. Of the remaining 198 specimens, 90 were positive by all three methods, 9 were positive by TOR and Max, and 3 were positive by TOR only. One negative specimen was initially inhibitory by Max. The remaining 95 specimens were negative by all methods. Toxigenic C. difficile culture was performed on the 12 discrepant samples. True C. difficile-positive status was defined as either positive by all three amplification assays or positive by toxigenic culture. Based on this definition, the sensitivity and specificity were 96.9% and 95% for Max and 92.8% and 100% for IMDx. In summary, both highly automated systems demonstrated excellent performance, and each has individual benefits, which will ensure that they will both have a niche in clinical laboratories.
Wilgen, Urs; Pretorius, Carel J; Gous, Rehna S; Martin, Cameron; Hale, Vincent J; Ungerer, Jacobus P J
Point-of-care testing for β-hCG has been widely advocated to allow rapid diagnosis/exclusion of pregnancy in the emergency department. A quantitative blood β-hCG assay has the additional benefit of being able to monitor the viability of pregnancy, using serial measurements, to determine the appropriate expected increase in β-hCG levels over time (e.g. ectopic pregnancy), and aiding in determining if an intrauterine gestational sac should be visible on sonographic imaging. Evaluation of the newly released Abbott i-STAT β-hCG point-of-care assay with the Beckman Coulter β-hCG laboratory assay in use. Whole blood, plasma and serum samples with a wide range of β-hCG concentrations were analysed by both methods. The Abbott I-STAT β-hCG compares favourably, can be performed on heparinised whole blood, plasma and serum, and shows acceptable accuracy and precision. However a hook effect at elevated β-hCG was shown in gestational trophoblastic disease as well as normal pregnancies. The i-STAT β-hCG performs acceptably in its intended use in the early detection of pregnancy, but results should always be interpreted within the clinical context, as a hook effect may occur. Crown Copyright © 2014. Published by Elsevier Inc. All rights reserved.
Poljak, Mario; Ostrbenk, Anja; Seme, Katja; Ucakar, Veronika; Hillemanns, Peter; Bokal, Eda Vrtacnik; Jancar, Nina; Klavs, Irena
The clinical performance of the Abbott RealTime High Risk HPV (human papillomavirus) test (RealTime) and that of the Hybrid Capture 2 HPV DNA test (hc2) were prospectively compared in the population-based cervical cancer screening setting. In women >30 years old (n = 3,129), the clinical sensitivity of RealTime for detection of cervical intraepithelial neoplasia of grade 2 (CIN2) or worse (38 cases) and its clinical specificity for lesions of less than CIN2 (3,091 controls) were 100% and 93.3%, respectively, and those of hc2 were 97.4% and 91.8%, respectively. A noninferiority score test showed that the clinical specificity (P < 0.0001) and clinical sensitivity (P = 0.011) of RealTime were noninferior to those of hc2 at the recommended thresholds of 98% and 90%. In the total study population (women 20 to 64 years old; n = 4,432; 57 cases, 4,375 controls), the clinical sensitivity and specificity of RealTime were 98.2% and 89.5%, and those of hc2 were 94.7% and 87.7%, respectively. The analytical sensitivity and analytical specificity of RealTime in detecting targeted HPV types evaluated with the largest sample collection to date (4,479 samples) were 94.8% and 99.8%, and those of hc2 were 93.4% and 97.8%, respectively. Excellent analytical agreement between the two assays was obtained (kappa value, 0.84), while the analytical accuracy of RealTime was significantly higher than that of hc2. RealTime demonstrated high intralaboratory reproducibility and interlaboratory agreement with 500 samples retested 61 to 226 days after initial testing in two different laboratories. RealTime can be considered to be a reliable and robust HPV assay clinically comparable to hc2 for the detection of CIN2+ lesions in a population-based cervical cancer screening setting.
Bailey, D; Martens, P; Mah, W; Yip, P M
To evaluate the performance of the Abbott ARCHITECT enzymatic assay for magnesium (3P68) in serum/plasma and urine against analytical goals based on biological variation. Analytical performance was evaluated according to CLSI protocols. Precision was examined using commercial chemistry controls. Accuracy was assessed against NIST SRM 956c, electrolytes in human serum. Correlation with the arsenazo Mg assay (7D70) was completed using patient samples (plasma, N = 101; urine, N = 90). Common interferences were examined in pooled patient specimens with high and low magnesium concentrations. The enzymatic Mg assay displayed imprecision of 1.7% at 0.72 mmol/L and 1.4% at 1.80 mmol/L (20 days, one calibration, one reagent lot). The linear range was verified between 0.18-7.0 mmol/L (plasma) and 0.01-10.69 mmol/L (urine). Results of the enzymatic assay (x) correlated well with the predicate assay (y) with the relationships y = 0.891x + 0.035, R = 0.967 (plasma) and y = 1.181x + 0.086, R = 0.997 (urine). Mean bias of the NIST SRM 956 c samples was -1.4%. This method showed minimal interference by hemoglobin (3g/L as hemolysate), lipemia (20 g/L Intralipid), unconjugated bilirubin (531 μmol/L), and ascorbate (680 μmol/L). The ARCHITECT Magnesium assay 3P68 achieved the desirable analytical quality specification of 4.8% for total allowable error. In comparison to the 7D70 assay, notable improvements are seen in precision, 30-day calibration stability, and minimal interference by hemolyzed and lipemic samples. © 2013.
Ausina, V; Gamboa, F; Gazapo, E; Manterola, J M; Lonca, J; Matas, L; Manzano, J R; Rodrigo, C; Cardona, P J; Padilla, E
Five hundred twenty processed respiratory specimens from 326 patients received for the diagnosis of tuberculosis or other mycobacterial infections were tested by means of the LCx Mycobacterium tuberculosis Assay from Abbott Laboratories, which uses ligase chain reaction technology for the direct detection of M. tuberculosis complex in respiratory specimens. The results of the LCx M. tuberculosis Assay were compared with the results of culture and staining techniques. After a combination of culture results and the patient's clinical data, a total of 195 specimens were collected from 110 patients who were positively diagnosed as having pulmonary tuberculosis. Twenty-three of these 195 specimens which corresponded to 10 patients with a history of pulmonary tuberculosis (TB) and anti-TB treatment ranging from 1 to 6 months were culture negative. The other 172 specimens were culture positive for M. tuberculosis. With an overall positivity rate of 37.5% (195 of 520 specimens), the sensitivity, specificity, and positive and negative predictive values were 90.8, 100, 100, and 94.7%, respectively, for the LCx M. tuberculosis Assay; 88.2, 100, 100, and 93.4%, respectively, for culture; and 82.6, 92, 72.9, and 97.6%, respectively, for acid-fast staining. For 161 specimens (82.6%) from patients smear positive for the disease and 34 specimens (17.4%) from patients smear negative for the disease, the sensitivity values for the LCx M. tuberculosis Assay were 98.8 and 53%, respectively. There were no statistically significant differences in the sensitivities and specificities between the LCx M. tuberculosis Assay and culture (P > 0.05). Conclusively, the LCx M. tuberculosis Assay has proved to have an acceptable sensitivity and a high specificity in detecting M. tuberculosis and has the potential of reducing the diagnosis time to an 8-h working day. PMID:9230369
Møller, Jens Kjølseth; Pedersen, Lisbeth Nørum; Persson, Kenneth
The clinical performance of two nucleic acid amplification assays targeting the cryptic plasmid and two assays targeting rRNA molecules in Chlamydia trachomatis was examined. First-catch urine samples from Malmoe, Sweden, were tested for C. trachomatis with the Abbott real-time PCR assay m2000 and an in-house PCR for the new variant strain of C. trachomatis with a deletion in the cryptic plasmid. Aliquots of the urine samples were sent to Aarhus, Denmark, and further examined with the Roche COBAS Amplicor CT (RCA) PCR, the Gen-Probe Aptima Combo 2 assay (AC2) targeting the C. trachomatis 23S rRNA, and the Aptima C. trachomatis assay (ACT) targeting the 16S rRNA molecule. A positive prevalence of 9% (163/1,808 urine samples examined) was detected according to the combined reference standard. The clinical sensitivity and specificity of the four assays were as follows: for ACT, 100% (163/163) and 99.9% (1,643/1,645), respectively; for AC2, 100% (163/163) and 99.6% (1,640/1,645); for m2000, 68.7% (112/163) and 99.9% (1,644/1,645); for RCA, 63.8% (104/163) and 99.9% (1,643/1,645). The two Gen-Probe assays detected all mutant strains characterized by the in-house PCR as having the deletion in the cryptic plasmid, whereas the Roche and the Abbott PCRs targeting the plasmid were both unable to detect the plasmid mutant. The difference in clinical sensitivity between the plasmid PCR assays m2000 and RCA, on the one hand, and the rRNA assays AC2 and ACT, on the other, could be attributed almost exclusively to the presence of the plasmid mutant in about one-quarter of the Chlamydia-positive samples examined.
Collinson, P O; Gaze, D; Goodacre, S
The aim of this study is to determine the imprecision profile, 99th percentile and diagnostic efficiency of a new high sensitivity cardiac troponin I (cTnI) assay. Total imprecision was assessed by following CLSI protocol EP15-A.14. Serum pools prepared from sera of known high cardiac troponin concentrations were adjusted by dilution with serum considered to be troponin free. Determination of the 99th-percentile reference value examined a fully characterized population that had undergone non-invasive cardiac imaging. Diagnostic accuracy utilised samples from the point of care arm of the RATPAC trial (Randomised Assessment of Treatment using Panel Assay of Cardiac markers), set in the emergency departments of six hospitals. Blood samples were taken on admission and 90min from admission. Diagnosis was based on the universal definition of myocardial infarction utilising laboratory measurements of cardiac troponin performed at the participating sites together with measurements performed in a core laboratory and compared by construction of receiver operator characteristic curves. Total imprecision was 4%-12.1% with 10% CV of 7ng/L. cTnI was measureable in 99.5% of the samples. Troponin values were influenced by gender but not by age. The 99th percentile was 14.8ng/L (18.1 males, 8.6 females). Progressive filtering of the population reduced the 99th percentile. For the diagnosis of MI on admission the area under the curve was 0.92, statistically indistinguishable from four other assays studied (0.90-0.94). The analytical performance of the new assay meets the criteria for a high sensitivity troponin assay. Copyright © 2014 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
Ceffa, Susanna; Luhanga, Richard; Andreotti, Mauro; Brambilla, Davide; Erba, Fulvio; Jere, Haswel; Mancinelli, Sandro; Giuliano, Marina; Palombi, Leonardo; Marazzi, Maria Cristina
Assessing treatment efficacy and early infant diagnosis (EID) are critical issues in HIV disease management. Point-of-care assays may greatly increase the possibility to access laboratory monitoring also in rural areas. Recently two new laboratory tests have been developed by Cepheid (Sunnyvale, California) the Xpert HIV-1 Viral Load for viral load determination and the Xpert HIV-1 Qualitative for early infant diagnosis. We conducted a study in Blantyre, Malawi, comparing the 2 methods versus the Abbott real time quantitative and qualitative assays, for viral load and EID respectively. We tested 300 plasma samples for viral load determination and 200 samples for infant diagnosis. HIV-1 RNA values of the 274 samples quantified by both assays were highly correlated (Pearson r=0.95, R(2)=0.90). In 90.9% of the cases the two methods were concordant in defining the HIV-1 RNA levels as detectable or undetectable. For EID, the Xpert HIV-1 Qualitative assay yielded the same identical results as the Abbott assay. Both the quantitative and the qualitative Xpert assays are promising tools to monitor treatment efficacy in HIV patients receiving treatment and for early diagnosis in HIV-exposed infants.
Ollier, Laurence; Laffont, Catherine; Kechkekian, Aurore; Doglio, Alain; Giordanengo, Valérie
Routine use of the automated chemiluminescent microparticle immunoassay Abbott ARCHITECT anti-HBc for diagnosis of hepatitis B is limited in case of borderline reactive sera with low signal close to the cut-off index. In order to determine the significance of anti-HBc detection when borderline reactivity occurs using the ARCHITECT anti-HBc assay, a comparative study was designed. 3540 serum samples collected over a 2-month period in the hospital of Nice were examined for markers of HBV infection (HBsAg, anti-HBs and anti-HBc). One hundred seven samples with sufficient volume and with borderline reactivity by the ARCHITECT assay were tested by two other anti-HBc assays, a microparticle enzyme immunoassay (MEIA, AxSYM Core, Abbott Laboratories, IL, USA) and an enzyme linked fluorescent assay (ELFA, VIDAS Anti-HBc Total II, bioMérieux, Lyon, France). Only 46 samples were confirmed by the AxSYM and the VIDAS assays. Additional serological information linked to patient history showed that the remaining samples (61) were false positives (11), had low titer of anti-HBc antibodies (13), or were inconclusive (37). This comparative study highlighted the existence of a grey zone around the cut-off index. Confirmative results through a different immunoassay are needed to confirm the diagnosis of HBV on borderline reactive sera using the ARCHITECT anti-HBc assay.
Khopkar, Priyanka; Mallav, Vikas; Chidrawar, Shweta; Kulkarni, Smita
The implementation of cost effective HIV-1 viral load assays in resource-limited settings have been an impediment for monitoring HIV-1 therapy. A study involving the comparative analytical performance of two HIV-1 viral load assays - Standard Roche COBAS(®) Amplicor™ HIV-1 Monitor(®) Test, version 1.5 (Roche Diagnostics, Basel, Switzerland) and Abbott HIV-1 RealTime™ assay (Abbott Molecular, Wiesbaden, Germany) was performed using 125 specimens in Pune, India. A strong correlation was observed between the manual endpoint reverse transcriptase polymerase chain reaction assay and the recent real time polymerase chain reaction assay (r=0.989, p value<0.0001) and agreement was 94.4%. Results of the study indicate a higher sensitivity of the Abbott HIV-1 RealTime™ assay for HIV-1 Virology Quality Assurance copy controls as compared to the Standard Roche COBAS(®) Amplicor™ HIV-1 Monitor(®) Test, version 1.5. Furthermore, features of the Abbott m2000rt RealTime™ PCR assay platform such as higher analytical sensitivity, automated/manual extraction platforms for high/low sample throughputs and ability to quantify a variety of infectious agents (Hepatitis B virus, Hepatitis C virus, Human Papillomavirus and Neisseria gonorrhoeae/Chlamydia trachomatis) justify its suitability in resource-limited Indian settings. Besides, the study also highlights utility of the precise Virology Quality Assurance validation template in performance evaluation of various quantitative viral load assays.
Sun, Hongye; Low, Karen E; Woo, Sam; Noble, Richard L; Graham, Ronald J; Connaughton, Sonia S; Gee, Melissa A; Lee, Linda G
We report a novel, real-time fluorogenic kinase assay. The peptide substrates are synthesized with a fluorescent dye and a hydrocarbon tail. The substrate self-assembles into micelles, increasing the local concentration of the dye and quenching its fluorescence. Upon phosphorylation, the fluorescence intensity increases 4-6-fold due to micelle reorganization. Both dynamic light scattering data and cryoelectron microscope images show that the size and the shape of the phosphopeptide micelles are significantly different from micelles of substrate peptide. The system provides a robust fluorescence increase in a real-time protein kinase assay. Unlike other fluorogenic systems, the fluorophore may be distant from the serine, threonine, or tyrosine that is phosphorylated. Assays for several kinases, including PKA, PKC, p38, MAPKAP K2, akt, Erk1, and src-family kinases, have been developed. IC(50) values of inhibitors for PKC betaII determined with this technology are consistent with published values. The utility of this assay to high-throughput screening was demonstrated with Sigma's LOPAC library, a collection of 640 compounds with known biological activities, and satisfactory results were obtained.
Peuchant, Olivia; de Diego, Sabrina; Le Roy, Chloé; Frantz-Blancpain, Sandrine; Hocké, Claude; Bébéar, Cécile; de Barbeyrac, Bertille
We compared 3 commercial real-time PCR assays, the Abbott RealTime CT/NG, the cobas® 4800 CT/NG, and the Cepheid Xpert® CT/NG, for the detection of Chlamydia trachomatis and Neisseria gonorrhoeae in vaginal swabs collected prospectively from pregnant women aged <25 years. The overall agreement among 2 assays ranged from 98.9% to 99.5% with a kappa score between 0.94 and 0.97 for C. trachomatis. For N. gonorrhoeae, the overall agreement was 100%. All kits allowed prompt and specific results for C. trachomatis and N. gonorrhoeae in young pregnant women.
Ndiege, Kenneth; Inzaule, Seth; Achieng, Rebecca; Williamson, John; Chih-Wei Chang, Joy; Ellenberger, Dennis; Nkengasong, John
Background Routine HIV viral load testing is not widely accessible in most resource-limited settings, including Kenya. To increase access to viral load testing, alternative sample types like dried blood spots (DBS), which overcome the logistic barriers associated with plasma separation and cold chain shipment need to be considered and evaluated. The current study evaluated matched dried blood spots (DBS) and dried plasma spots (DPS) against plasma using the Abbott M 2000 (Abbott) and Roche Cobas Ampliprep/Cobas TaqMan (CAP/CTM) quantitative viral load assays in western Kenya. Methods Matched plasma DBS and DPS were obtained from 200 HIV-1 infected antiretroviral treatment (ART)-experienced patients attending patient support centers in Western Kenya. Standard quantitative assay performance parameters with accompanying 95% confidence intervals (CI) were assessed at the assays lower detection limit (400cps/ml for CAP/CTM and 550cps/ml for Abbott) using SAS version 9.2. Receiver operating curves (ROC) were further used to assess viral-load thresholds with best assay performance (reference assay CAP/CTM plasma). Results Using the Abbott test, the sensitivity and specificity, respectively, for DPS were (97.3%, [95%CI: 93.2–99.2] and 98.1% [95%CI: 89.7–100]) and those for DBS (93.9% [95%CI: 88.8–97.2] and 88.0% [95%CI: 82.2–92.4]). The correlation and agreement using paired plasma and DPS/DBS were strong, with r2 = 90.5 and rc = 68.1. The Bland-Altman relative percent change was 95.3 for DPS, (95%CI: 90.4–97.7) and 73.6 (95%CI: 51.6–86.5) for DBS. Using the CAP/CTM assay, the sensitivity for DBS was significantly higher compared to DPS (100.0% [95% CI: 97.6–100.0] vs. 94.7% [95%CI: 89.8–97.7]), while the specificity for DBS was lower: 4%, [95% CI: 0.4–13.7] compared to DPS: 94.0%, [95% CI: 83.5–98.7]. When compared under different clinical relevant thresholds, the accuracy for the Abbott assay was 95% at the 1000cps/ml cut-off with a sensitivity and
Cook, Linda; Sullivan, KaWing; Krantz, Elizabeth M; Bagabag, Arthur; Jerome, Keith R
A variety of methods have been used to determine hepatitis C virus (HCV) genotypes. Because therapeutic decisions for chronic HCV-related hepatitis are made on the basis of genotype, it is important that genotype be accurately determined by clinical laboratories. Existing methods are often subjective, inaccurate, manual, time-consuming, and contamination prone. We therefore evaluated real-time reverse transcription-PCR (RT-PCR) reagents that have recently become commercially available (Abbott HCV Genotype ASR). The assay developed by our laboratory starts with purified RNA and can be performed in 4 to 5 h. An initial evaluation of 479 samples was done with a restriction fragment length polymorphism (RFLP) method and the RT-PCR assay, and discrepant samples were sequenced. An additional 1,200 samples were then tested, and data from all assays were used to evaluate the efficiency and specificity of each genotype-specific reaction. Good correlation between results by the two methods was seen. Discrepant samples included those indeterminate by the RT-PCR assay (n = 110) and a subset that were incorrectly called 2a by the RFLP method (n = 75). The real-time RT-PCR assay performed well with genotype 1, 2, and 3 samples. Inadequate numbers of samples were available to evaluate fully genotypes 4, 5, and 6. Analysis of each primer-probe set demonstrated that weak cross-reactive amplifications were common but usually did not interfere with the genotype determination. However, in about 1% of samples, two or more genotypes amplified at roughly equivalent amounts. Further studies are necessary to determine whether these mixed-genotype samples are true mixtures or a reflection of occasional cross-reactive amplifications.
Roe, M; Kishiyama, C; Davidson, K; Schaefer, L; Todd, J
We directly compared three techniques for the diagnosis of group A streptococcal pharyngitis in 500 symptomatic children seen in the Emergency Department or Child Care Clinic of The Children's Hospital of Denver. Throats were vigorously swabbed with two rayon swabs, which were transported immediately to the Microbiology Laboratory. Each swab was cultured aerobically on Strep A Isolation Agar (Remel) and then tested for antigen-one swab by the Strep A OIA optical immune assay (BioStar) and the other by the TestPack Plus Strep A (Abbott) technique. Each test was performed blind to the others. The refrigerated pledget was cultured in Todd-Hewitt broth if an antigen test was positive and both direct plate cultures were negative (the "gold standard" was any culture positive). All isolates were serologically grouped. Of 500 complete patient cultures, 151 (30%) were positive for group A streptococcal growth. The two antigen tests gave comparable results with an average sensitivity of 83%. Each was significantly (P < 0.02) less sensitive than its corresponding culture. The BioStar Strep A OIA optical immune assay produced significantly (P < 0.003) more false-positive results than did the Abbott test. Rapid antigen testing is not sensitive enough to eliminate the need for backup cultures. PMID:7650184
Capece, Mark C.; Kornberg, Guy L.; Petrov, Alexey
A high-throughput assay for real-time measurement of translation rates in cell-free protein synthesis (SNAP assay) is described. The SNAP assay enables quantitative, real-time measurement of overall translation rates in vitro via the synthesis of O6-alkylguanine DNA O6-alkyltransferase (SNAP). SNAP production is continuously detected by fluorescence produced by the reaction of SNAP with a range of quenched fluorogenic substrates. The capabilities of the assay are exemplified by measurements of the activities of Escherichia coli MRE600 ribosomes and fluorescently labeled E. coli mutant ribosomes in the PURExpress translation system and by determination of the 50% inhibitory concentrations (IC50) of three common macrolide antibiotics. PMID:25525154
CLSI-based transference of the CALIPER database of pediatric reference intervals from Abbott to Beckman, Ortho, Roche and Siemens Clinical Chemistry Assays: direct validation using reference samples from the CALIPER cohort.
Estey, Mathew P; Cohen, Ashley H; Colantonio, David A; Chan, Man Khun; Marvasti, Tina Binesh; Randell, Edward; Delvin, Edgard; Cousineau, Jocelyne; Grey, Vijaylaxmi; Greenway, Donald; Meng, Qing H; Jung, Benjamin; Bhuiyan, Jalaluddin; Seccombe, David; Adeli, Khosrow
The CALIPER program recently established a comprehensive database of age- and sex-stratified pediatric reference intervals for 40 biochemical markers. However, this database was only directly applicable for Abbott ARCHITECT assays. We therefore sought to expand the scope of this database to biochemical assays from other major manufacturers, allowing for a much wider application of the CALIPER database. Based on CLSI C28-A3 and EP9-A2 guidelines, CALIPER reference intervals were transferred (using specific statistical criteria) to assays performed on four other commonly used clinical chemistry platforms including Beckman Coulter DxC800, Ortho Vitros 5600, Roche Cobas 6000, and Siemens Vista 1500. The resulting reference intervals were subjected to a thorough validation using 100 reference specimens (healthy community children and adolescents) from the CALIPER bio-bank, and all testing centers participated in an external quality assessment (EQA) evaluation. In general, the transferred pediatric reference intervals were similar to those established in our previous study. However, assay-specific differences in reference limits were observed for many analytes, and in some instances were considerable. The results of the EQA evaluation generally mimicked the similarities and differences in reference limits among the five manufacturers' assays. In addition, the majority of transferred reference intervals were validated through the analysis of CALIPER reference samples. This study greatly extends the utility of the CALIPER reference interval database which is now directly applicable for assays performed on five major analytical platforms in clinical use, and should permit the worldwide application of CALIPER pediatric reference intervals. Copyright © 2013 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
Utilizing the phenomenon of nucleases exposing oligonucleotide phosphate backbones to phosphatases we present a novel quantitative method for kinetics of nuclease catalysis. Inorganic phosphate released from nuclease products by phosphatases could be quantified in real-time by a fluorescent sensor of inorganic phosphate. Two different nucleases were employed, showing the versatility of this assay for multiple turnover label-free nuclease studies. PMID:27101307
Quinn, Frank A; Scopp, Richard; Lach, Agnes; Drake, Christopher; Mo, May; Albright, John; Trimpe, Kevin
Important performance characteristics for any thyroid-stimulating hormone (TSH) assay include low-end sensitivity, precision across a wide dynamic range, and linear specimen dilution. Laboratories often characterize the performance of their TSH assay using many different sample matrices (e.g. native human serum, synthetic buffer, processed human serum, etc.). However, this can lead to possible confusion as the relationships between sample matrix and assay performance are often poorly understood. The purpose of our study was to evaluate the performance of the ARCHITECT i2000 TSH assay using a variety of different sample matrices. Functional sensitivity was found to be essentially equivalent in both hyperthyroid patient sera (0.0035 microIU/ml) and TSH affinity-stripped (0.0038 microIU/ml) sample matrices. Assay imprecision was also independent of sample matrix, with total imprecision < or = 5.3% for both synthetic buffer and serum matrices. Similarly, specimens were found to dilute linearly over a wide range in both serum and synthetic buffer matrices. We conclude that the i2000 TSH assay measures TSH similarly in a variety of sample matrices. This is an important design feature for this immunoassay which provides assurance that analytical performance assessed with matrices other than unprocessed human serum are representative of assay performance seen with patient specimens.
Comparative evaluation of three automated systems for DNA extraction in conjunction with three commercially available real-time PCR assays for quantitation of plasma Cytomegalovirus DNAemia in allogeneic stem cell transplant recipients.
Bravo, Dayana; Clari, María Ángeles; Costa, Elisa; Muñoz-Cobo, Beatriz; Solano, Carlos; José Remigia, María; Navarro, David
Limited data are available on the performance of different automated extraction platforms and commercially available quantitative real-time PCR (QRT-PCR) methods for the quantitation of cytomegalovirus (CMV) DNA in plasma. We compared the performance characteristics of the Abbott mSample preparation system DNA kit on the m24 SP instrument (Abbott), the High Pure viral nucleic acid kit on the COBAS AmpliPrep system (Roche), and the EZ1 Virus 2.0 kit on the BioRobot EZ1 extraction platform (Qiagen) coupled with the Abbott CMV PCR kit, the LightCycler CMV Quant kit (Roche), and the Q-CMV complete kit (Nanogen), for both plasma specimens from allogeneic stem cell transplant (Allo-SCT) recipients (n = 42) and the OptiQuant CMV DNA panel (AcroMetrix). The EZ1 system displayed the highest extraction efficiency over a wide range of CMV plasma DNA loads, followed by the m24 and the AmpliPrep methods. The Nanogen PCR assay yielded higher mean CMV plasma DNA values than the Abbott and the Roche PCR assays, regardless of the platform used for DNA extraction. Overall, the effects of the extraction method and the QRT-PCR used on CMV plasma DNA load measurements were less pronounced for specimens with high CMV DNA content (>10,000 copies/ml). The performance characteristics of the extraction methods and QRT-PCR assays evaluated herein for clinical samples were extensible at cell-based standards from AcroMetrix. In conclusion, different automated systems are not equally efficient for CMV DNA extraction from plasma specimens, and the plasma CMV DNA loads measured by commercially available QRT-PCRs can differ significantly. The above findings should be taken into consideration for the establishment of cutoff values for the initiation or cessation of preemptive antiviral therapies and for the interpretation of data from clinical studies in the Allo-SCT setting.
Comparative Evaluation of Three Automated Systems for DNA Extraction in Conjunction with Three Commercially Available Real-Time PCR Assays for Quantitation of Plasma Cytomegalovirus DNAemia in Allogeneic Stem Cell Transplant Recipients▿
Bravo, Dayana; Clari, María Ángeles; Costa, Elisa; Muñoz-Cobo, Beatriz; Solano, Carlos; José Remigia, María; Navarro, David
Limited data are available on the performance of different automated extraction platforms and commercially available quantitative real-time PCR (QRT-PCR) methods for the quantitation of cytomegalovirus (CMV) DNA in plasma. We compared the performance characteristics of the Abbott mSample preparation system DNA kit on the m24 SP instrument (Abbott), the High Pure viral nucleic acid kit on the COBAS AmpliPrep system (Roche), and the EZ1 Virus 2.0 kit on the BioRobot EZ1 extraction platform (Qiagen) coupled with the Abbott CMV PCR kit, the LightCycler CMV Quant kit (Roche), and the Q-CMV complete kit (Nanogen), for both plasma specimens from allogeneic stem cell transplant (Allo-SCT) recipients (n = 42) and the OptiQuant CMV DNA panel (AcroMetrix). The EZ1 system displayed the highest extraction efficiency over a wide range of CMV plasma DNA loads, followed by the m24 and the AmpliPrep methods. The Nanogen PCR assay yielded higher mean CMV plasma DNA values than the Abbott and the Roche PCR assays, regardless of the platform used for DNA extraction. Overall, the effects of the extraction method and the QRT-PCR used on CMV plasma DNA load measurements were less pronounced for specimens with high CMV DNA content (>10,000 copies/ml). The performance characteristics of the extraction methods and QRT-PCR assays evaluated herein for clinical samples were extensible at cell-based standards from AcroMetrix. In conclusion, different automated systems are not equally efficient for CMV DNA extraction from plasma specimens, and the plasma CMV DNA loads measured by commercially available QRT-PCRs can differ significantly. The above findings should be taken into consideration for the establishment of cutoff values for the initiation or cessation of preemptive antiviral therapies and for the interpretation of data from clinical studies in the Allo-SCT setting. PMID:21697323
De Keukeleire, Steven; Desmet, Stefanie; Lagrou, Katrien; Oosterlynck, Julie; Verhulst, Manon; Van Besien, Jessica; Saegeman, Veroniek; Reynders, Marijke
The performance of Elecsys Syphilis was compared to Architect Syphilis TP and Reformulated Architect Syphilis TP. The overall sensitivity and specificity were 98.4% and 99.5%, 97.7% and 97.1%, and 99.2% and 99.7% respectively. The assays are comparable and considered adequate for syphilis screening.
Kim, Seung Min; Jeong, Yoojung; Sohn, Jang Wook; Kim, Min Ja
Legionella pneumophila serogroup 1 (sg1) accounts for the majority of infections in humans, but other Legionella species are also associated with human disease. In this study, a new SYBR Green I-based multiplex real-time PCR assay in a single reaction was developed to allow the rapid detection and differentiation of Legionella species by targeting specific gene sequences. Candidate target genes were selected, and primer sets were designed by referring to comparative genomic hybridization data of Legionella species. The Legionella species-specific groES primer set successfully detected all 30 Legionella strains tested. The xcpX and rfbA primers specifically detected L. pneumophila sg1-15 and L. pneumophila sg1, respectively. In addition, this assay was validated by testing clinical samples and isolates. In conclusion, this novel multiplex real-time PCR assay might be a useful diagnostic tool for the rapid detection and differentiation of Legionella species in both clinical and epidemiological studies.
Henterich, Nadine; Osman, Awad A; Méndez, Enrique; Mothes, Thomas
Patients with coeliac disease (gluten-sensitive enteropathy) are intolerant against gliadins from wheat and the respective proteins from related cereals and have to keep a lifelong gluten-free diet. For control of gliadin in gluten-free food sensitive assay techniques are necessary. We developed an immunopolymerase chain reaction (iPCR) assay for gliadin. In this technique immunological detection of gliadin by a monoclonal antibody R5 conjugated with an oligonucleotide is amplified by PCR. For quantification, iPCR was performed as real-time PCR (real-time iPCR) in one step. By means of real-time iPCR, the sensitivity of gliadin analysis was increased more than 30-fold above the level reached by enzyme immunoassay. Real time-iPCR using R5 directly conjugated with oligonucleotide was clearly more sensitive than real time-iPCR applying sequentially biotinylated R5, streptavidin, and biotinylated oligonucleotide. With directly conjugated R5 gliadin was detected at a concentration as low as 0.16 ng/mL corresponding to 16 microg gliadin/100 g food or 0.16 ppm (corresponding to 0.25 g of food extracted in 10 mL of solvent and 25-fold dilution of the extract prior to analysis). This is the first report applying the highly sensitive technique of iPCR for gliadin analysis. Furthermore, this is the first approach to perform real-time iPCR in one step without changing the reaction vessels after enzyme immunoassay for subsequent PCR analysis thus minimizing risks of contamination and loss of sensitivity.
Quantification of DNA in Plasma by an Automated Real-Time PCR Assay (Cytomegalovirus PCR Kit) for Surveillance of Active Cytomegalovirus Infection and Guidance of Preemptive Therapy for Allogeneic Hematopoietic Stem Cell Transplant Recipients▿
Gimeno, Concepción; Solano, Carlos; Latorre, José C.; Hernández-Boluda, Juan C.; Clari, María A.; Remigia, María J.; Furió, Santiago; Calabuig, Marisa; Tormo, Nuria; Navarro, David
The performance of a plasma real-time PCR (cytomegalovirus [CMV] PCR kit; Abbott Diagnostics) was compared with that of the antigenemia assay for the surveillance of active CMV infection in 42 allogeneic hematopoietic stem cell transplantation (Allo-SCT) recipients. A total of 1,156 samples were analyzed by the two assays. Concordance between the two assays was 82.2%. Plasma DNA levels correlated with the number of pp65-positive cells, particularly prior to the initiation of preemptive therapy. Fifty-seven episodes of active CMV infection were detected in 37 patients: 18 were defined solely by the PCR assay and four were defined on the basis of the antigenemia assay. Either a cutoff of 288 CMV DNA copies/ml or a 2.42-log10 increase of DNAemia levels between two consecutive PCR positive samples was an optimal value to discriminate between patients requiring preemptive therapy and those not requiring therapy on the basis of the antigenemia results. The real-time PCR assay allowed an earlier diagnosis of active CMV infection and was a more reliable marker of successful clearance of CMV from the blood. Analysis of the kinetics of DNAemia levels at a median of 7 days posttreatment allowed the prediction of the response to CMV therapy. Two patients developed CMV colitis. The PCR assay tested positive both before the onset of symptoms and during the disease period. The plasma real-time PCR from Abbott is more suitable than the antigenemia assay for monitoring active CMV infection in Allo-SCT recipients and may be used for guiding preemptive therapy in this clinical setting. PMID:18753357
Chevaliez, Stéphane; Dubernet, Fabienne; Dauvillier, Claude; Hézode, Christophe; Pawlotsky, Jean-Michel
Sensitive and accurate hepatitis C virus (HCV) RNA detection and quantification is essential for the management of chronic hepatitis C therapy. Currently available platforms and assays are usually batched and require at least 5hours of work to complete the analyses. The aim of this study was to evaluate the ability of the newly developed Aptima HCV Quant Dx assay that eliminates the need for batch processing and automates all aspects of nucleic acid testing in a single step, to accurately detect and quantify HCV RNA in a large series of patients infected with different HCV genotypes. The limit of detection was estimated to be 2.3 IU/mL. The specificity of the assay was 98.6% (95% confidence interval: 96.1%-99.5%). Intra-assay and inter-assay coefficients of variation ranged from 0.09% to 5.61%, and 1.05% to 3.65%, respectively. The study of serum specimens from patients infected with HCV genotypes 1 to 6 showed a satisfactory relationship between HCV RNA levels measured by the Aptima HCV Quant Dx assay, and both real-time PCR comparators (Abbott RealTime HCV and Cobas AmpliPrep/Cobas TaqMan HCV Test, version 2.0, assays). the new Aptima HCV Quant Dx assay is rapid, sensitive, reasonably specific and reproducible and accurately quantifies HCV RNA in serum samples from patients with chronic HCV infection, including patients on antiviral treatment. The Aptima HCV Quant Dx assay can thus be confidently used to detect and quantify HCV RNA in both clinical trials with new anti-HCV drugs and clinical practice in Europe and the US. Copyright © 2017 Elsevier B.V. All rights reserved.
Chevaliez, Stéphane; Dauvillier, Claude; Dubernet, Fabienne; Poveda, Jean-Dominique; Laperche, Syria; Hézode, Christophe; Pawlotsky, Jean-Michel
Sensitive and accurate hepatitis B virus (HBV) DNA detection and quantification are essential to diagnose HBV infection, establish the prognosis of HBV-related liver disease, and guide the decision to treat and monitor the virological response to antiviral treatment and the emergence of resistance. Currently available HBV DNA platforms and assays are generally designed for batching multiple specimens within an individual run and require at least one full day of work to complete the analyses. The aim of this study was to evaluate the ability of the newly developed, fully automated, one-step Aptima HBV Quant assay to accurately detect and quantify HBV DNA in a large series of patients infected with different HBV genotypes. The limit of detection of the assay was estimated to be 4.5 IU/ml. The specificity of the assay was 100%. Intra-assay and interassay coefficients of variation ranged from 0.29% to 5.07% and 4.90% to 6.85%, respectively. HBV DNA levels from patients infected with HBV genotypes A to F measured with the Aptima HBV Quant assay strongly correlated with those measured by two commercial real-time PCR comparators (Cobas AmpliPrep/Cobas TaqMan HBV test, version 2.0, and Abbott RealTime HBV test). In conclusion, the Aptima HBV Quant assay is sensitive, specific, and reproducible and accurately quantifies HBV DNA in plasma samples from patients with chronic HBV infections of all genotypes, including patients on antiviral treatment with nucleoside or nucleotide analogues. The Aptima HBV Quant assay can thus confidently be used to detect and quantify HBV DNA in both clinical trials with new anti-HBV drugs and clinical practice. Copyright © 2017 American Society for Microbiology.
Inoue, Takako; Hmwe, Su Su; Shimada, Noritomo; Kato, Keizo; Ide, Tatsuya; Torimura, Takuji; Kumada, Takashi; Toyoda, Hidenori; Tsubota, Akihito; Takaguchi, Koichi; Wakita, Takaji; Tanaka, Yasuhito
The aim of this study was to determine the efficacy of two hepatitis C virus (HCV) real-time PCR assays, the COBAS AmpliPrep/COBAS TaqMan HCV test (CAP/CTM) and the Abbott RealTime HCV test (ART), for predicting the clinical outcomes of patients infected with HCV who received telaprevir (TVR)-based triple therapy or daclatasvir/asunaprevir (DCV/ASV) dual therapy. The rapid virological response rates in patients receiving TVR-based triple therapy were 92% (23/25) and 40% (10/25) for CAP/CTM and ART, respectively. The false omission rate (FOR) of ART was 93.3% (14/15), indicating that CAP/CTM could accurately predict clinical outcome in the early phase. In an independent examination of 20 patients receiving TVR-based triple therapy who developed viral breakthrough or relapse, the times to HCV disappearance by ART were longer than by CAP/CTM, whereas the times to HCV reappearance were similar. In an independent experiment of WHO standard HCV RNA serially diluted in serum containing TVR, the analytical sensitivities of CAP/CTM and ART were similar. However, cell cultures transfected with HCV and grown in medium containing TVR demonstrated that ART detected HCV RNA for a longer time than CAP/CTM. Similar results were found for 42 patients receiving DCV/ASV dual therapy. The FOR of ART was 73.3% (11/15) at week 8 after initiation of therapy, indicating that ART at week 8 could not accurately predict the clinical outcome. In conclusion, although CAP/CTM and ART detected HCV RNA with comparable analytical sensitivity, CAP/CTM might be preferable for predicting the clinical outcomes of patients receiving protease inhibitor-based therapy. PMID:28118381
Theodore, M. Jordan; Mair, Raydel; Trujillo-Lopez, Elizabeth; du Plessis, Mignon; Wolter, Nicole; Baughman, Andrew L.; Hatcher, Cynthia; Vuong, Jeni; Lott, Lisa; von Gottberg, Anne; Sacchi, Claudio; McDonald, J. Matthew; Messonnier, Nancy E.; Mayer, Leonard W.
Neisseria meningitidis, Haemophilus influenzae, and Streptococcus pneumoniae are important causes of meningitis and other infections, and rapid, sensitive, and specific laboratory assays are critical for effective public health interventions. Singleplex real-time PCR assays have been developed to detect N. meningitidis ctrA, H. influenzae hpd, and S. pneumoniae lytA and serogroup-specific genes in the cap locus for N. meningitidis serogroups A, B, C, W135, X, and Y. However, the assay sensitivity for serogroups B, W135, and Y is low. We aimed to improve assay sensitivity and develop multiplex assays to reduce time and cost. New singleplex real-time PCR assays for serogroup B synD, W135 synG, and Y synF showed 100% specificity for detecting N. meningitidis species, with high sensitivity (serogroup B synD, 99% [75/76]; W135 synG, 97% [38/39]; and Y synF, 100% [66/66]). The lower limits of detection (LLD) were 9, 43, and 10 copies/reaction for serogroup B synD, W135 synG, and Y synF assays, respectively, a significant improvement compared to results for the previous singleplex assays. We developed three multiplex real-time PCR assays for detection of (i) N. meningitidis ctrA, H. influenzae hpd, and S. pneumoniae lytA (NHS assay); (ii) N. meningitidis serogroups A, W135, and X (AWX assay); and (iii) N. meningitidis serogroups B, C, and Y (BCY assay). Each multiplex assay was 100% specific for detecting its target organisms or serogroups, and the LLD was similar to that for the singleplex assay. Pairwise comparison of real-time PCR between multiplex and singleplex assays showed that cycle threshold values of the multiplex assay were similar to those for the singleplex assay. There were no substantial differences in sensitivity and specificity between these multiplex and singleplex real-time PCR assays. PMID:22170919
Yoo, Ju Eun; Lee, Cheonghoon; Park, SungJun; Ko, GwangPyo
Human noroviruses are widespread and contagious viruses causing nonbacterial gastroenteritis. Real-time reverse transcription quantitative PCR (real-time RT-qPCR) is currently the gold standard for sensitive and accurate detection for these pathogens and serves as a critical tool in outbreak prevention and control. Different surveillance teams, however, may use different assays and variability in specimen conditions may lead to disagreement in results. Furthermore, the norovirus genome is highly variable and continuously evolving. These issues necessitate the re-examination of the real-time RT-qPCR's robustness in the context of accurate detection as well as the investigation of practical strategies to enhance assay performance. Four widely referenced real-time RT-qPCR assays (Assay A-D) were simultaneously performed to evaluate characteristics such as PCR efficiency, detection limit, as well as sensitivity and specificity with RT-PCR, and to assess the most accurate method for detecting norovirus genogroups I and II. Overall, Assay D was evaluated to be the most precise and accurate assay in this study. A Zen internal quencher, which decreases nonspecific fluorescence during the PCR reaction, was added to Assay D's probe which further improved assay performance. This study compared several detection assays for noroviruses and an improvement strategy based on such comparisons provided useful characterizations of a highly optimized real-time RT-qPCR assay for norovirus detection.
Abd El Galil, Khaled H.; El Sokkary, M. A.; Kheira, S. M.; Salazar, Andre M.; Yates, Marylynn V.; Chen, Wilfred; Mulchandani, Ashok
A nucleic acid sequence-based amplification (NASBA) assay in combination with a molecular beacon was developed for the real-time detection and quantification of hepatitis A virus (HAV). A 202-bp, highly conserved 5′ noncoding region of HAV was targeted. The sensitivity of the real-time NASBA assay was tested with 10-fold dilutions of viral RNA, and a detection limit of 1 PFU was obtained. The specificity of the assay was demonstrated by testing with other environmental pathogens and indicator microorganisms, with only HAV positively identified. When combined with immunomagnetic separation, the NASBA assay successfully detected as few as 10 PFU from seeded lake water samples. Due to its isothermal nature, its speed, and its similar sensitivity compared to the real-time RT-PCR assay, this newly reported real-time NASBA method will have broad applications for the rapid detection of HAV in contaminated food or water. PMID:16269748
Gubala, Aneta J.; Proll, David F.
A multiplex real-time PCR assay was developed using molecular beacons for the detection of Vibrio cholerae by targeting four important virulence and regulatory genes. The specificity and sensitivity of this assay, when tested with pure culture and spiked environmental water samples, were high, surpassing those of currently published PCR assays for the detection of this organism. PMID:16957277
agents in the blood stream the diseases are also difficult to diagnose by laboratory methods. For that reason we have developed real - time PCR assays to...detect rickettsial disease agents both at the genus and the species level. Real - time PCR assays were developed to identify: 1) pathogenic Rickettsia...calculate the sensitivity of the assays. These real - time PCR assays were found to be capable of detecting rickettsial disease agents quickly and with great sensitivity and specificity.
Vincart, Benoit; De Mendonça, Ricardo; Rottiers, Sylvianne; Vermeulen, Françoise; Struelens, Marc J; Denis, Olivier
A novel real-time PCR (RT-PCR) assay was developed for detection of Bordetella pertussis in respiratory specimens by targeting the pertactin gene. In vitro evaluation with reference strains and quality control samples showed analytical sensitivity equivalent to and specificity superior to those of PCR assays which target the IS481 element. The pertactin-based RT-PCR assay offers better discrimination between B. pertussis and other Bordetella species than previously described assays.
Vilcek, Stefan; Vlasakova, Michaela; Jackova, Anna
Light Upon eXtension real-time PCR (LUX real-time PCR) assay was developed for the detection of porcine circovirus type 2 (PCV2). The primers flanking a 114 bp fragment were selected from ORF1. The optimized assay could detect 20 viral copies of pBluescript SK+ plasmid containing inserted PCV2 DNA. The dynamic range of quantitative analysis covered a 7-order interval ranging from 20 to 2 x 10(8) genome equivalents per assay with the best results in the range from 2 x 10(2) to 2 x 10(7) viral copies. The LUX real-time PCR assay had a high specificity since it detected PCV2 but not PCV1, CSFV, PRRSV or negative samples. There was good agreement between the LUX real-time PCR and the conventional PCR when lymph nodes from PCV2 infected animals were tested. A comparison of the LUX real-time PCR with the TaqMan PCR and SYBR Green PCR indicated that the amount of viral copies determined using linear calibration curve differed from assay to assay but not more than an order. LUX real-time PCR, similar to the TaqMan PCR, was more specific for generation of fluorogenic signal than SYBR Green PCR.
This paper explores Maude Abbott's internationally significant career in medicine and her parallel commitment to caring for her sister, Alice Abbott. An examination of Abbott's life reveals the difficulties faced by an ambitious Canadian woman in medicine from the 1890s to the 1920s; difficulties compounded by caring for a sister with a mental illness. The Abbott archive suggests that it was far more difficult for a woman doctor to make the kind of sharp distinction between public and private life that might be expected of professional men.
Traditional plating methods are reliable means for Campylobacter identification from poultry samples but automated gene-based detection systems now available can reduce assay time, data collection and analysis. Bio-Rad and DuPont Qualicon recently introduced Campylobacter assays for their real-time ...
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...
Berg, T; Tesoriero, L; Hailstones, D L
To develop a sensitive real-time PCR-based protocol for the detection of Xanthomonas campestris pathovars from Brassica seed. A 5' nuclease real-time PCR assay was developed to screen Brassica spp. seed for the presence of X. campestris pathovars that cause black rot. The assay amplifies a 78-bp segment of the X. campestris hrpF gene and a 100-bp segment of the Brassica spp. 18S-25S internal transcribed spacer region. The Brassica spp. target provides an internal control for the amplification process to prevent false negatives that may arise from inhibitors that are often present in extracts from plant material. Whilst the primers were compatible with SYBR Green I assays, the use of fluorescently labelled probes in a 5' nuclease assay afforded greatest sensitivity and specificity. Seed batches carrying one artificially infected seed among 10,000 were readily detected using the assay. The multiplex real-time PCR assay permitted the rapid detection of pathogenic strains of X. campestris from bacterial colonies, Brassica seed and plants. Strains of X. campestris pathogenic to brassicas were readily detected from seed via a multiplex 5' nuclease real-time PCR assay. The real-time assay offers an improvement in sensitivity and a reduced turn-around time over the conventional multiplex PCR. Real-time PCR can be used to rapidly screen Brassica spp. seed batches for the presence of X. campestris pathovars. This assay provides a means for growers and the seed industry to be aware of the black rot status of their planting material, so that they may more effectively employ disease control measures or seed disinfection.
Ekins, Jason; Peters, Sharla M; Jones, Yolanda L; Swaim, Heidi; Ha, Tai; La Neve, Fabio; Civera, Tiziana; Blackstone, George; Vickery, Michael C L; Marion, Bill; Myers, Michael J; Yancy, Haile F
The U.S. Food and Drug Administration (FDA) has previously validated a real-time PCR-based assay that is currently being used by the FDA and several state laboratories as the official screening method. Due to several shortcomings to the assay, a multiplex real-time PCR assay (MRTA) to detect three ruminant species (bovine, caprine, and ovine) was developed using a lyophilized bead design. The assay contained two primer or probe sets: a "ruminant" set to detect bovine-, caprine-, and ovine-derived materials and a second set to serve as an internal PCR control, formatted using a lyophilized bead design. Performance of the assay was evaluated against stringent acceptance criteria developed by the FDA's Center for Veterinary Medicine's Office of Research. The MRTA for the detection of ruminant DNA passed the stringent acceptance criteria for specificity, sensitivity, and selectivity. The assay met sensitivity and reproducibility requirements by detecting 30 of 30 complete feed samples fortified with meals at 0.1 % (wt/wt) rendered material from each of the three ruminant species. The MRTA demonstrated 100 % selectivity (0.0 % false positives) for negative controls throughout the assessment period. The assay showed ruggedness in both sample selection and reagent preparation. Second and third analyst trials confirmed the quality of the written standard operating procedure with consistency of results. An external laboratory participating in a peer-verification trial demonstrated 100 % specificity in identifying bovine meat and bone meal, while exhibiting a 0.03 % rate of false positives. The assay demonstrated equal levels of sensitivity and reproducibility compared with the FDA's current validated real-time PCR assay. The assay detected three prohibited species in less than 1.5 h of total assay time, a significant improvement over the current real-time assay. These results demonstrated this assay's suitability for routine regulatory use both as a primary screening tool
Hänsel, C.; Mertens, K.; Elschner, M. C.; Melzer, F.
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
Arikan, S; Rex, J H
November 1998, Aronex signed a licensing collaboration with Abbott Laboratories for the worldwide rights to nystatin LF .
Liu, Chao; Chang, Le; Jia, Tingting; Guo, Fei; Zhang, Lu; Ji, Huimin; Zhao, Junpeng; Wang, Lunan
Quantification Hepatitis B virus (HBV) DNA plays a critical role in the management of chronic HBV infections. However, HBV is a DNA virus with high levels of genetic variation, and drug-resistant mutations have emerged with the use of antiviral drugs. If a mutation caused a sequence mismatched in the primer or probe of a commercial DNA quantification kit, this would lead to an underestimation of the viral load of the sample. The aim of this study was to determine whether commercial kits, which use only one pair of primers and a single probe, accurately quantify the HBV DNA levels and to develop an improved duplex real-time PCR assay. We developed a new duplex real-time PCR assay that used two pairs of primers and two probes based on the conserved S and C regions of the HBV genome. We performed HBV DNA quantitative detection of HBV samples and compared the results of our duplex real-time PCR assays with the COBAS TaqMan HBV Test version 2 and Daan real-time PCR assays. The target region of the discordant sample was amplified, sequenced, and validated using plasmid. The results of the duplex real-time PCR were in good accordance with the commercial COBAS TaqMan HBV Test version 2 and Daan real-time PCR assays. We showed that two samples from Chinese HBV infections underestimated viral loads when quantified by the Roche kit because of a mismatch between the viral sequence and the reverse primer of the Roche kit. The HBV DNA levels of six samples were undervalued by duplex real-time PCR assays of the C region because of mutations in the primer of C region. We developed a new duplex real-time PCR assay, and the results of this assay were similar to the results of commercial kits. The HBV DNA level could be undervalued when using the COBAS TaqMan HBV Test version 2 for Chinese HBV infections owing to a mismatch with the primer/probe. A duplex real-time PCR assay based on the S and C regions could solve this problem to some extent.
Sofi Ibrahim, M; Kulesh, David A; Saleh, Sharron S; Damon, Inger K; Esposito, Joseph J; Schmaljohn, Alan L; Jahrling, Peter B
We developed a highly sensitive and specific assay for the rapid detection of smallpox virus DNA on both the Smart Cycler and LightCycler platforms. The assay is based on TaqMan chemistry with the orthopoxvirus hemagglutinin gene used as the target sequence. With genomic DNA purified from variola virus Bangladesh 1975, the limit of detection was estimated to be approximately 25 copies on both machines. The assay was evaluated in a blinded study with 322 coded samples that included genomic DNA from 48 different isolates of variola virus; 25 different strains and isolates of camelpox, cowpox, ectromelia, gerbilpox, herpes, monkeypox, myxoma, rabbitpox, raccoonpox, skunkpox, vaccinia, and varicella-zoster viruses; and two rickettsial species at concentrations mostly ranging from 100 fg/ microl to 1 ng/ microl. Contained within those 322 samples were variola virus DNA, obtained from purified viral preparations, at concentrations of 1 fg/ microl to 1 ng/ microl. On the Smart Cycler platform, 2 samples with false-positive results were detected among the 116 samples not containing variola virus tested; i.e., the overall specificity of the assay was 98.3%. On the LightCycler platform, five samples with false-positive results were detected (overall specificity, 95.7%). Of the 206 samples that contained variola virus DNA ranging in concentrations from 100 fg/ microl to 1 ng/ microl, 8 samples were considered negative on the Smart Cycler platform and 1 sample was considered negative on the LightCycler platform. Thus, the clinical sensitivities were 96.1% for the Smart Cycler instrument and 99.5% for the LightCycler instrument. The vast majority of these samples were derived from virus-infected cell cultures and variola virus-infected tissues; thus, the DNA material contained both viral DNA and cellular DNA. Of the 43 samples that contained purified variola virus DNA ranging in concentration from 1 fg/ microl to 1 ng/ microl, the assay correctly detected the virus in all 43
Sofi Ibrahim, M.; Kulesh, David A.; Saleh, Sharron S.; Damon, Inger K.; Esposito, Joseph J.; Schmaljohn, Alan L.; Jahrling, Peter B.
We developed a highly sensitive and specific assay for the rapid detection of smallpox virus DNA on both the Smart Cycler and LightCycler platforms. The assay is based on TaqMan chemistry with the orthopoxvirus hemagglutinin gene used as the target sequence. With genomic DNA purified from variola virus Bangladesh 1975, the limit of detection was estimated to be approximately 25 copies on both machines. The assay was evaluated in a blinded study with 322 coded samples that included genomic DNA from 48 different isolates of variola virus; 25 different strains and isolates of camelpox, cowpox, ectromelia, gerbilpox, herpes, monkeypox, myxoma, rabbitpox, raccoonpox, skunkpox, vaccinia, and varicella-zoster viruses; and two rickettsial species at concentrations mostly ranging from 100 fg/μl to 1 ng/μl. Contained within those 322 samples were variola virus DNA, obtained from purified viral preparations, at concentrations of 1 fg/μl to 1 ng/μl. On the Smart Cycler platform, 2 samples with false-positive results were detected among the 116 samples not containing variola virus tested; i.e., the overall specificity of the assay was 98.3%. On the LightCycler platform, five samples with false-positive results were detected (overall specificity, 95.7%). Of the 206 samples that contained variola virus DNA ranging in concentrations from 100 fg/μl to 1 ng/μl, 8 samples were considered negative on the Smart Cycler platform and 1 sample was considered negative on the LightCycler platform. Thus, the clinical sensitivities were 96.1% for the Smart Cycler instrument and 99.5% for the LightCycler instrument. The vast majority of these samples were derived from virus-infected cell cultures and variola virus-infected tissues; thus, the DNA material contained both viral DNA and cellular DNA. Of the 43 samples that contained purified variola virus DNA ranging in concentration from 1 fg/μl to 1 ng/μl, the assay correctly detected the virus in all 43 samples on both the Smart Cycler
Ogawa, Eiichi; Furusyo, Norihiro; Murata, Masayuki; Toyoda, Kazuhiro; Eiraku, Kunimitsu; Shimizu, Motohiro; Harada, Yuji; Mitsumoto, Fujiko; Takayama, Koji; Okada, Kyoko; Kainuma, Mosaburo; Hayashi, Jun
Monitoring hepatitis C virus (HCV) kinetics during antiviral treatment is recommended for determining the best form of treatment management. We compared the measurement of HCV RNA by two Real-time PCR assays during the first 12weeks phase of telaprevir in combination with pegylated interferon α2b and ribavirin treatment for chronic hepatitis C patients. The viral kinetics of 65 patients with HCV genotype 1b was assessed. HCV RNA was tested at baseline, on day 3, and every week from 1 to 12 by both the first-generation Roche COBAS® AmpliPrep/COBAS® TaqMan® HCV (CAP/CTM) assay and the Abbott RealTime HCV (ART) assay. A total of 910 serum samples were obtained from the 65 patients. Of these, 168 (28.5%) of the 590 samples HCV RNA negative by CAP/CTM were positive by ART. In contrast, 17 (3.9%) of the 439 samples HCV RNA negative by ART were positive by CAP/CTM. The rates of HCV RNA negativity by ART at weeks 3, 4, and 5 were significantly lower than those by CAP/CTM (21.5% vs. 50.8%, 36.9% vs. 70.8% and 44.6% vs. 81.5%; P<0.001, P<0.0001 and P<0.05, respectively). Although the ART is superior for the determination of HCV RNA negativity, the predictive value of detectable HCV RNA for non-sustained virological response (non-SVR) by CAP/CTM is higher than by ART at weeks 4, 6, and 8. We also found that 16 (24.6%) by CAP/CTM and 28 (43.1%) by ART had a reappearance of residual HCV RNA during the telaprevir treatment period. However, the reappearance of residual HCV RNA was not associated with non-SVR. In conclusion, a significant difference was found between the two real-time PCR assays for the assessment of virological response based on undetectable HCV RNA.
Bortoletto, Gladis; Campagnolo, Davide; Mirandola, Silvia; Comastri, Giuseppe; Severini, Letizia; Pulvirenti, Franco Renato; Alberti, Alfredo
Antiviral therapy for chronic hepatitis C may cause transient on-treatment response followed by post-treatment relapse. We have compared the prognostic value for post-treatment relapse of minimal hepatitis C residual viraemia detected at end-of-therapy by transcription mediated assay (TMA) and by Abbott RealTime PCR HCV assay. Minimal residual viraemia was investigated in 202 HCV patients who had completed a full course of Pegylated Interferon (PEG-IFN) plus ribavirin and were HCV-RNA negative by conventional PCR in two separate serum samples obtained during the last week of therapy and the results were then correlated with post-treatment outcome. Minimal residual viraemia was detected in 22/202 (11%, 95% CI: 7-16%) and in 28/202 (13.8%, 95% CI 10-19%) patients by TMA and by Abbott RealTime HCV assay, respectively, with 92% concordant results. Post-treatment relapse was seen in 81.8% (95% CI: 60-93%) of TMA positive and in 82.1% (95% CI: 64-92%) of Abbott RealTime HCV assay positive cases compared to 16.6% (95% CI: 12-23%) of TMA negative and 17.2% (95% CI: 12-23%) of Abbott RealTime HCV assay negative patients. These results indicate that TMA and the Abbott RealTime HCV assay have comparable sensitivity and specificity in identifying minimal residual viraemia at the end of antiviral therapy, with excellent predictive value for post-treatment relapse. Copyright © 2010 Elsevier B.V. All rights reserved.
Quantitation of estrogen receptor in seventy-five specimens of breast cancer: comparison between an immunoassay (Abbott ER-EIA monoclonal) and a (3H)estradiol binding assay based on isoelectric focusing in polyacrylamide gel
Pousette, A.; Gustafsson, S.A.; Thoernblad, A.M.N.; Nordgren, A.; Saellstroem, J.Li.; Lindgren, A.; Sundelin, P.; Gustafsson, J.A.
Quantitation of estrogen receptor has been performed in cytosol prepared from 75 specimens of breast cancer tissue from patients who had not received hormonal therapy. The study was performed in order to compare an immunoassay (Abbott Laboratories, North Chicago, IL) with our currently used method for estrogen receptor analysis based on isoelectric focusing of (/sup 3/H)estradiol-receptor complex in polyacrylamide gels. Using linear regression analysis, a regression coefficient (slope) of 1.30 and a correlation coefficient of 0.75 were calculated. The differences in results between the two methods are probably partly explained by the fact that the ligand-based method only measures unoccupied receptor, whereas the immunoassay detects the total amount of receptor, resulting in generally slightly higher concentrations with the latter method. However, in five of 75 specimens the ligand-based method gave a considerably higher concentration of estrogen receptor. This was most probably explained by partial proteolysis resulting in the formation of receptor fragment(s), which was undetectable with the immunoassay but detectable with the ligand-based method. These observations underline the importance of careful handling of specimens during the whole immunoassay procedure.
Shin, Bo-Moon; Yoo, Sun Mee; Shin, Won Chang
We evaluated the performance of four commercial nucleic acid amplification tests (NAATs: Xpert C. difficile, BD MAX Cdiff, IMDx C. difficile for Abbott m2000, and Illumigene C. difficile) for direct and rapid detection of Clostridium difficile toxin genes. We compared four NAATs on the same set of 339 stool specimens (303 prospective and 36 retrospective specimens) with toxigenic culture (TC). Concordance rate among four NAATs was 90.3% (306/339). Based on TC results, the sensitivity and specificity were 90.0% and 92.9% for Xpert; 86.3% and 89.3% for Max; 84.3% and 94.4% for IMDx; and 82.4% and 93.7% for Illumigene, respectively. For 306 concordant cases, there were 11 TC-negative/NAATs co-positive cases and 6 TC-positive/NAATs co-negative cases. Among 33 discordant cases, 18 were only single positive in each NAAT (Xpert, 1; Max, 12; IMDx, 1; Illumigene, 4). Positivity rates of the four NAATs were associated with those of semi-quantitative cultures, which were maximized in grade 3 (>100 colony-forming unit [CFU]) compared with grade 1 (<10 CFU). Commercial NAATs may be rapid and reliable methods for direct detection of tcdA and/or tcdB in stool specimens compared with TC. Some differences in the sensitivity of the NAATs may partly depend on the number of toxigenic C. difficile in stool specimens.
Karlsson, Malin; Wallensten, Anders; Lundkvist, Ake; Olsen, Björn; Brytting, Maria
A screening system including a new real-time PCR assay for the monitoring of influenza A virus in wild birds was developed. The real-time PCR assay uses SYBR green chemistry and the primers are targeting the matrix gene of influenza A virus. The performance of the assay was compared with two other assays, one assay also using SYBR green chemistry and one assay using TaqMan chemistry, i.e. a specific probe. A total of 45 fecal bird samples were analysed for influenza A virus in three different PCR reactions. Overall, 26 samples were positive in at least one of the three real-time PCR assays. Of the 26 samples, 18 were positive by all three reactions. Eight samples were found positive exclusively by the two SYBR green reactions, six of which were detected by both SYBR green reactions. Of the 26 positive samples, 15 samples were verified as positive either by virus isolation or influenza A M2-gene PCR. The results showed that the two SYBR green systems had a higher performance regarding the detection of influenza A as compared to the PCR reaction using a specific probe.
Wu, Xuping; Wang, Jianfang; Song, Jinyun; Li, Jiayan; Yang, Yongfeng
Hepatitis C virus (HCV) is one of the common reasons of liver fibrosis and hepatocellular carcinoma (HCC). Early, rapid and accurate HCV RNA detection is important to prevent and control liver disease. A simultaneous amplification and testing (SAT) assay, which is based on isothermal amplification of RNA and real-time fluorescence detection, was designed to optimize routine HCV RNA detection. In this study, HCV RNA and an internal control (IC) were amplified and analyzed simultaneously by SAT assay and detection of fluorescence using routine real-time PCR equipment. The assay detected as few as 10 copies of HCV RNA transcripts. We tested 705 serum samples with SAT, among which 96.4% (680/705) showed consistent results compared with routine real-time PCR. About 92% (23/25) discordant samples were confirmed to be same results as SAT-HCV by using a second real-time PCR. The sensitivity and specificity of SAT-HCV assay were 99.6% (461/463) and 100% (242/242), respectively. In conclusion, the SAT assay is an accurate test with a high specificity and sensitivity which may increase the detection rate of HCV. It is therefore a promising tool to diagnose HCV infection.
Orosz, Erika; Perkátai, Katalin; Kapusinszky, Beatrix; Farkas, Agnes; Kucsera, István
Simple real-time PCR assay with one set of primer and probe for rapid, sensitive qualitative and quantitative detection of Entamoeba histolytica has been used. Consensus sequences were used to amplify a species-specific region of the 16S rRNA gene, and fluorescence resonance energy transfer hybridization probes were used for detection in a LightCycler platform (Roche). The anchor probe sequence was designed to be a perfect match for the 16S rRNA gene of Entamoeba species, while the acceptor probe sequence was designed for Entamoeba histolytica, which allowed differentiation. The performed characteristics of the real-time PCR assay were compared with ELISA antigen and microscopical detection from 77 samples of individuals with suspected clinical diagnosis of imported E. histolytica infection. Stool and liver abscess pus samples were examined with analytical sensitivity of 5 parasites per PCR reaction. The melting curve means Tms (standard deviation) in clinical isolates were 54°C. The real-time assay was 100% sensitive and specific for differentiation of Entamoeba histolytica, compared with conventional ELISA or microscopy. This real-time PCR assay with melting curve analysis is rapid, and specific for the detection and differentiation of Entamoeba histolytica. The suitability for routine use of this assay in clinical diagnostic laboratories is discussed.
Leś, Katarzyna; Przybylski, Maciej; Dzieciatkowski, Tomasz; Młynarczyk, Grazyna
Infections with human enteroviruses are common worldwide and cause a wide range of signs and symptoms. Nowadays in current diagnostics procedures older virological methods, such virus isolation in a cell cultures and seroneutralisation assay, are replaced with molecular biology tests. The aim of the study was development of real-time PCR assay for detection of human adenoviruses. DNA isolated from MK2 cell line infected with nineteen different enterovirus strains was used for development of a qualitative real-time PCR assay using primers targeting a conserved region of the 5'UTR region and a specific TaqMan probe. The analytical sensitivity of real-time PCR assay was tested using serial dilutions of Coxackie A9 cDNA in range between 10 degrees and 10(-8). For comparison typical end-point detected RT-PCR for enterovirus detection with the same cDNA dilutions was made. The sensitivity of novel method was about ten thousand-fold higher than older one. The conclusion is that real-time PCR is very advisable in diagnostics of diseases caused with enteroviruses. The high level of sensitivity, specificity, accuracy, and rapidity provided by this assay are favorable for the use in the detection of enteroviral RNA in clinical specimens, especially from neuroinfections.
Else, Elizabeth A; Swoyer, Ryan; Zhang, Yuhua; Taddeo, Frank J; Bryan, Janine T; Lawson, John; Van Hyfte, Inez; Roberts, Christine C
Real-time type-specific multiplex human papillomavirus (HPV) PCR assays were developed to detect HPV DNA in samples collected for the efficacy determination of the quadrivalent HPV (type 6, 11, 16, and 18) L1 virus-like particle (VLP) vaccine (Gardasil). Additional multiplex (L1, E6, and E7 open reading frame [ORF]) or duplex (E6 and E7 ORF) HPV PCR assays were developed to detect high-risk HPV types, including HPV type 31 (HPV31), HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, and HPV59. Here, we evaluated clinical specimen concordance and compared the limits of detection (LODs) between multiplex HPV PCR assays and the INNO-LiPA HPV Genotyping Extra assay, which detects 28 types, for the 14 HPV types common to both of these methods. Overall HPV detection agreement rates were >90% for swabs and >95% for thin sections. Statistically significant differences in detection were observed for HPV6, HPV16, HPV18, HPV35, HPV39, HPV45, HPV56, HPV58, and HPV59 in swabs and for HPV45, HPV58, and HPV59 in thin sections. Where P was <0.05, discordance was due to detection of more HPV-positive samples by the multiplex HPV PCR assays. LODs were similar for eight HPV types, significantly lower in multiplex assays for five HPV types, and lower in INNO-LiPA for HPV6 only. LODs were under 50 copies for all HPV types, with the exception of HPV39, HPV58, and HPV59 in the INNO-LiPA assay. The overall percent agreement for detection of 14 HPV types between the type-specific multiplex HPV PCR and INNO-LiPA genotyping assays was good. The differences in positive sample detection favored multiplex HPV PCR, suggesting increased sensitivity of HPV DNA detection by type-specific multiplex HPV PCR assays.
James, Mindy; Blagden, Trenna; Moncrief, Ian; Burans, James P; Schneider, Katherine; Fletcher, Jacqueline
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.
33672 Bacillus megaterium ................................................................ NA...Assay for a Unique Chromosomal Sequence of Bacillus anthracis Elizabeth Bode,1 William Hurtle,2† and David Norwood1* United States Army Medical...modification 4 June 2004/Accepted 9 August 2004 Real-time PCR has become an important method for the rapid identification of Bacillus anthracis since the
Wojewoda, Christina M; Spahlinger, Timothy; Harmon, Marlene Louise; Schnellinger, Brian; Li, Qing; Dejelo, Corazon; Schmotzer, Christine; Zhou, Lan
Viral load monitoring of HIV-1 has become standard of care in HIV-1 positive patients. In this study, we evaluated the performance characteristics of the Roche Cobas AmpliPrep/Cobas TaqMan HIV-1 test version 2.0 (CAP/CTM v2.0) in comparison with Roche Cobas AmpliPrep/Cobas TaqMan HIV-1 test version 1.0 (CAP/CTM v1.0) and Abbott RealTime HIV-1 assay (m2000), with special emphasis on the quantitation of clinically controversial low-level viral loads. The performance characteristics of CAP/CTM v2.0 were confirmed by the validation study. All three assays performed comparably, with Abbott m2000 showing slightly decreased sensitivity for detection of viral loads close to the lower limit of quantitation. Follow-up of patients with low-level viral loads revealed that some of those represent single viral blips; however, a significant portion of these patients have intermittent or persistent low-positive viremia. We conclude that CAP/CTM v2.0 is an accurate and reliable assay for HIV-1 viral load monitoring.
Kirstein, Shelli L; Atienza, Josephine M; Xi, Biao; Zhu, Jenny; Yu, Naichen; Wang, Xiaobo; Xu, Xiao; Abassi, Yama A
In this paper we have explored the utility of the real-time cell electronic sensing (RTCES, ACEA Biosciences Inc., San Diego, CA) system for monitoring the quality of live cells in cell-based assays as well as for assay development. We have demonstrated that each cell type displays unique growth kinetic profiles that provide a quantitative account of cell behavior and can be used as a diagnostic tool for cellular quality control. The utility of the specific signature patterns was shown by demonstrating the significant differences in primary cell behavior depending on the supplier. In addition, the RT-CES system was able to differentiate cell behavior depending on the passage stage of the cells. The utility of the RT-CES system as an assay development tool was demonstrated in cytotoxicity assays. The RT-CES system not only provides information regarding the potency of cytotoxic compounds, but in addition relates potency to the rate of the response for each concentration of the compound tested, which is important for understanding the mechanism of compound action. Moreover, real-time display of cytotoxicity data by the RT-CES system allows for calculation of real-time 50% inhibitory concentration (IC50) values or determination of optimal IC(50) value. In summary, the RT-CES system provides high content and information-rich data that are beyond the scope of single-point assays.
Mohn, Stein Christian; Ulvik, Arve; Jureen, Roland; Willems, Rob J L; Top, Janetta; Leavis, Helen; Harthug, Stig; Langeland, Nina
Rapid and accurate identification of carriers of resistant microorganisms is an important aspect of efficient infection control in hospitals. Traditional identification methods of antibiotic-resistant bacteria usually take at least 3 to 4 days after sampling. A duplex real-time PCR assay was developed for rapid detection of ampicillin-resistant Enterococcus faecium (ARE). Primers and probes that are used in this assay specifically detected the D-Ala-D-Ala ligase gene of E. faecium and the modified penicillin-binding protein 5 gene (pbp5) carrying the Glu-to-Val substitution at position 629 (Val-629) in a set of 129 tested E. faecium strains with known pbp5 sequence. Presence of the Val-629 in the strain set from 11 different countries was highly correlated with ampicillin resistance. In a screening of hospitalized patients, the real-time PCR assay yielded a sensitivity and a specificity for the detection of ARE colonization of 95% and 100%, respectively. The results were obtained 4 h after samples were harvested from overnight broth of rectal swab samples, identifying both species and the resistance marker mutation in pbp5. This novel assay reliably identifies ARE 2 to 3 days more quickly than traditional culture methods, thereby increasing laboratory throughput, making it useful for rectal screening of ARE. The assay demonstrates the advantages of real-time PCR for detection of nosocomial pathogens.
Tizolova, Anette; Brun, Delphine; Guiso, Nicole; Guillot, Sophie
Bordetella parapertussis is a causative agent of whooping cough in humans, and B. bronchiseptica is causing wide variety of respiratory infections in mammals, including humans. Specific diagnostic tests are not currently available. Our first objective was to develop a real-time PCR test for the specific detection of B. bronchiseptica based on the previously described end-point PCR, targeting an intergenomic sequence of the fla gene locus, but it has not been reached. However, there is cross-reactivity between B. parapertussis and B. bronchiseptica. Therefore, the targeted region of several clinical isolates of both species was sequenced, and alignment of the sequences allowed the development of a 2-step real-time PCR assay. The first PCR assay detected the DNA of all clinical isolates of both B. bronchiseptica and B. parapertussis tested. The second PCR assay detected only the DNA of B. parapertussis clinical isolates, thereby allowing discrimination between B. parapertussis and B. bronchiseptica.
Kato, Cecilia Y; Chung, Ida H; Robinson, Lauren K; Austin, Amy L; Dasch, Gregory A; Massung, Robert F
Two novel real-time PCR assays were developed for the detection of Rickettsia spp. One assay detects all tested Rickettsia spp.; the other is specific for Rickettsia rickettsii. Evaluation using DNA from human blood and tissue samples showed both assays to be more sensitive than nested PCR assays currently in use at the CDC.
Zhou, Wenhui; Meisenheimer, Poncho; Vidugiris, Gediminas; Cali, James J.; Gautam, Prson; Wennerberg, Krister; Vidugiriene, Jolanta
Abstract Real-time continuous monitoring of cellular processes offers distinct advantages over traditional endpoint assays. A comprehensive representation of the changes occurring in live cells over the entire length of an experiment provides information about the biological status of the cell and informs decisions about the timing of treatments or the use of other functional endpoint assays. We describe a homogeneous, nonlytic, bioluminescent assay that measures cell viability in real time. This time-dependent measurement allowed us to monitor cell health for 72 h from the same test samples, distinguish differential cell growth, and investigate drug mechanism of action by analyzing time- and dose-dependent drug effects. The real-time measurements also allowed us to detect cell death immediately (>75% signal decrease within 15 min of digitonin addition), analyze drug potency versus efficacy, and identify cytostatic versus toxic drug effects. We screened an oncology compound library (Z′ = 0.7) and identified compounds with varying activity at different time points (1.6% of the library showed activity within 3 h, whereas 35.4% showed a response by 47 h). The assay compared well with orthogonal endpoint cell viability assays and additionally provided data at multiple time points and the opportunity to multiplex assays on the same cells. To test the advantage of time-dependent measurements to direct optimal timing of downstream applications, we used the real-time cell viability assay to determine the ideal time to measure caspase activity by monitoring the onset of cell death and multiplexing a luminescent caspase activation assay on the same test samples. PMID:26383544
Duellman, Sarah J; Zhou, Wenhui; Meisenheimer, Poncho; Vidugiris, Gediminas; Cali, James J; Gautam, Prson; Wennerberg, Krister; Vidugiriene, Jolanta
Real-time continuous monitoring of cellular processes offers distinct advantages over traditional endpoint assays. A comprehensive representation of the changes occurring in live cells over the entire length of an experiment provides information about the biological status of the cell and informs decisions about the timing of treatments or the use of other functional endpoint assays. We describe a homogeneous, nonlytic, bioluminescent assay that measures cell viability in real time. This time-dependent measurement allowed us to monitor cell health for 72 h from the same test samples, distinguish differential cell growth, and investigate drug mechanism of action by analyzing time- and dose-dependent drug effects. The real-time measurements also allowed us to detect cell death immediately (>75% signal decrease within 15 min of digitonin addition), analyze drug potency versus efficacy, and identify cytostatic versus toxic drug effects. We screened an oncology compound library (Z' = 0.7) and identified compounds with varying activity at different time points (1.6% of the library showed activity within 3 h, whereas 35.4% showed a response by 47 h). The assay compared well with orthogonal endpoint cell viability assays and additionally provided data at multiple time points and the opportunity to multiplex assays on the same cells. To test the advantage of time-dependent measurements to direct optimal timing of downstream applications, we used the real-time cell viability assay to determine the ideal time to measure caspase activity by monitoring the onset of cell death and multiplexing a luminescent caspase activation assay on the same test samples.
Tatti, Kathleen M; Sparks, Kansas N; Boney, Kathryn O; Tondella, Maria Lucia
A novel multitarget real-time PCR (RT-PCR) assay for the rapid identification of Bordetella pertussis, B. parapertussis, and B. holmesii was developed using multicopy insertion sequences (ISs) in combination with the pertussis toxin subunit S1 (ptxS1) singleplex assay. The RT-PCR targets for the multiplex assay include IS481, commonly found in B. pertussis and B. holmesii; IS1001 of B. parapertussis; and the IS1001-like sequence of B. holmesii. Overall, 402 Bordetella species and 66 non-Bordetella species isolates were tested in the multitarget assay. Cross-reactivity was found only with 5 B. bronchiseptica isolates, which were positive with IS1001 of B. parapertussis. The lower limit of detection (LLOD) of the multiplex assay was similar to the LLOD of each target in an individual assay format, which was approximately 1 genomic equivalent per reaction for all targets. A total of 197 human clinical specimens obtained during cough-illness outbreak investigations were used to evaluate the multitarget RT-PCR assay. The multiplex assay results from 87 clinical specimens were compared to the individual RT-PCR assay and culture results. The multitarget assay is useful as a diagnostic tool to confirm B. pertussis infections and to rapidly identify other Bordetella species. In conclusion, the use of this multitarget RT-PCR approach increases specificity, while it decreases the amount of time, reagents, and specimen necessary for RT-PCRs used for accurate diagnosis of pertussis-like illness.
Gabitzsch, Elizabeth S; Vera-Tudela, Rommelle; Eisen, Rebecca J; Bearden, Scott W; Gage, Kenneth L; Zeidner, Nordin S
A real-time quantitative polymerase chain reaction (qPCR) assay was developed for Yersina pestis. The qPCR assay was developed utilizing a conserved region of the Y. pestis ferric iron uptake regulator gene (fur) to design primers and a fluorescent (FAM-labeled) TaqMan probe. The assay was optimized using cultured Y. pestis (UG05-0454) and was confirmed to work with strains from 3 Y. pestis biovars. The optimized assay was capable of detecting a single organism of cultured Y. pestis and as little as 300 bacteria in infected flea triturates. This qPCR assay enables rapid enumeration of Y. pestis bacterium in laboratory-infected fleas when compared with conventional serial dilution plating.
Albini, Sarah; Sigrist, Brigitte; Güttinger, Regula; Schelling, Claude; Hoop, Richard K; Vögtlin, Andrea
To aid in the rapid diagnosis of myxomatosis in rabbits, a real-time polymerase chain reaction (PCR) for the specific detection of Myxoma virus is described. Primers and probe were designed to amplify a 147-bp fragment within the Serp2 gene. The assay was able to detect 23 copies of a synthesized oligo indicating a reliable sensitivity. In addition, the real-time PCR did not detect the Rabbit fibroma virus used in myxomatosis vaccines. The novel PCR was shown to be able to detect Myxoma virus in fresh and paraffin-embedded rabbit tissues originating from myxomatosis cases from various regions in Switzerland.
Lal-Nag, Madhu; McGee, Lauren; Titus, Steven A; Brimacombe, Kyle; Michael, Sam; Sittampalam, Gurusingham; Ferrer, Marc
Two-dimensional monolayer cell proliferation assays for cancer drug discovery have made the implementation of large-scale screens feasible but only seem to reflect a simplified view that oncogenes or tumor suppressor genes are the genetic drivers of cancer cell proliferation. However, there is now increased evidence that the cellular and physiological context in which these oncogenic events occur play a key role in how they drive tumor growth in vivo and, therefore, in how tumors respond to drug treatments. In vitro 3D spheroid tumor models are being developed to better mimic the physiology of tumors in vivo, in an attempt to improve the predictability and efficiency of drug discovery for the treatment of cancer. Here we describe the establishment of a real-time 3D spheroid growth, 384-well screening assay. The cells used in this study constitutively expressed green fluorescent protein (GFP), which enabled the real-time monitoring of spheroid formation and the effect of chemotherapeutic agents on spheroid size at different time points of sphere growth and drug treatment. This real-time 3D spheroid assay platform represents a first step toward the replication in vitro of drug dosing regimens being investigated in vivo. We hope that further development of this assay platform will allow the investigation of drug dosing regimens, efficacy, and resistance before preclinical and clinical studies.
Bode, Elizabeth; Hurtle, William; Norwood, David
Real-time PCR has become an important method for the rapid identification of Bacillus anthracis since the 2001 anthrax mailings. Most real-time PCR assays for B. anthracis have been developed to detect virulence genes located on the pXO1 and pXO2 plasmids. In contrast, only two published chromosomal targets exist, the rpoB gene and the gyrA gene. In the present study, subtraction-hybridization with a plasmid-cured B. anthracis tester strain and a Bacillus cereus driver was used to find a unique chromosomal sequence. By targeting this region, a real-time assay was developed with the Ruggedized Advanced Pathogen Identification Device. Further testing has revealed that the assay has 100% sensitivity and 100% specificity, with a limit of detection of 50 fg of DNA. The results of a search for sequences with homology with the BLAST program demonstrated significant alignment to the recently published B. anthracis Ames strain, while an inquiry for protein sequence similarities indicated homology with an abhydrolase from B. anthracis strain A2012. The importance of this chromosomal assay will be to verify the presence of B. anthracis independently of plasmid occurrence. PMID:15583318
Rupprom, Kitwadee; Chavalitshewinkoon-Petmitr, Porntip; Diraphat, Pornphan; Kittigul, Leera
Noroviruses are the leading cause of acute gastroenteritis in humans. Real-time reverse transcription-polymerase chain reaction (real-time RT-PCR) is a promising molecular method for the detection of noroviruses. In this study, the performance of three TaqMan real-time RT-PCR assays was assessed, which were one commercially available real-time RT-PCR kit (assay A: Norovirus Real Time RT-PCR kit) and two in-house real-time RT-PCR assays (assay B: LightCycler RNA Master Hybprobe and assay C: RealTime ready RNA Virus Master). Assays A and B showed higher sensitivity than assay C for norovirus GI, while they all had the same sensitivity (10(3) DNA copies/mL) for GII DNA standard controls. Assay B had the highest efficiency for both genogroups. No cross-reactivity was observed among GI and GII noroviruses, rotavirus, hepatitis A virus, and poliovirus. The detection rates of these assays in GI and GII norovirus-positive fecal samples were not significantly different. However, the mean quantification cycle (Cq) value of assay B for GII was lower than assays A and C with statistical significance (P-value, 0.000). All three real-time RT-PCR assays could detect a variety of noroviruses including GI.2, GII.2, GII.3, GII.4, GII.6, GII.12, GII.17, and GII.21. This study suggests assay B as a suitable assay for the detection and quantification of noroviruses GI and GII due to good analytical sensitivity and higher performance to amplify norovirus on DNA standard controls and clinical samples.
Our earlier glycoproteomic studies have identified bisecting glycoslyation and core fucosylation changes on particular glycoproteins in endometrioid ovarian cancer tissues and plasma (Abbott et al, 2010, Proteomics). We have validated that these glycan changes occur on the same glycoproteins in serous ovarian cancer plasma using a lectin-pull down western blot assays. We would like to used pooled reference samples to develop a sensitive magnetic bead-based assay to detect these glycoproteins with bisecting and core fucosylation changes.
Lanotte, Philippe; Plouzeau, Chloé; Burucoa, Christophe; Grélaud, Carole; Guillot, Sophie; Guiso, Nicole; Garnier, Fabien
We evaluated the performances of 4 commercial real-time PCR kits for Bordetella pertussis IS481 sequence detection in nasopharyngeal aspirates by comparison with an in-house real-time PCR assay. Among them, the Simplexa Bordetella pertussis/parapertussis assay (Focus Diagnostics), the SmartCycler Bordetella pertussis/parapertussis assay (Cepheid), and Bordetella R-gene (Argene) present sensitivities over 90%. One kit proved unsuitable for routine clinical use. PMID:21918018
Thomas, Luke; Stat, Michael; Evans, Richard D.; Kennington, W. Jason
Pocillopora damicornis is one of the most extensively studied coral species globally, but high levels of phenotypic plasticity within the genus make species identification based on morphology alone unreliable. As a result, there is a compelling need to develop cheap and time-effective molecular techniques capable of accurately distinguishing P. damicornis from other congeneric species. Here, we develop a fluorescence-based quantitative real-time PCR (qPCR) assay to genotype a single nucleotide polymorphism that accurately distinguishes P. damicornis from other morphologically similar Pocillopora species. We trial the assay across colonies representing multiple Pocillopora species and then apply the assay to screen samples of Pocillopora spp. collected at regional scales along the coastline of Western Australia. This assay offers a cheap and time-effective alternative to Sanger sequencing and has broad applications including studies on gene flow, dispersal, recruitment and physiological thresholds of P. damicornis.
Barr, N B; Ledezma, L A; Farris, R E; Epstein, M E; Gilligan, T M
A molecular assay for diagnosis of light brown apple moth, Epiphyas postvittana (Walker) (Lepidoptera: Tortricidae), in North America is reported. The assay multiplexes two TaqMan real-time polymerase chain reaction (RT-PCR) probe systems that are designed to target DNA segments of the internal transcribed spacer region 2 (ITS2) and 18S rRNA gene. The RT-PCR probe designed for the 18S target recognizes a DNA sequence conserved in all of the moths included in the study and functions as a control in the assay. The second probe recognizes a segment of the ITS2 specifically found in E. postvittana and not found in the other moths included in the study, i.e., this segment is not conserved. Inclusion of the two markers in a single multiplex reaction did not affect assay performance. The assay was tested against 637 moths representing > 90 taxa in 15 tribes in all three subfamilies in the Tortricidae. The assay generated no false negatives based on analysis of 355 E. postvittana collected from California, Hawaii, England, New Zealand, and Australia. Analysis of a data set including 282 moths representing 41 genera generated no false positives. Only three inconclusive results were generated from the 637 samples. Spike experiments demonstrated that DNA contamination in the assay can affect samples differently. Contaminated samples analyzed with the ITS2 RT-PCR assay and DNA barcode methodology by using the cytochrome oxidase I gene can generate contradictory diagnoses.
Wiczk, Justyna; Westphal, Kinga; Rak, Janusz
Real-time PCR (qPCR) - a modern methodology primarily used for studying gene expression has been employed for the quantitative assay of an important class of DNA damage - single strand breaks. These DNA lesions which may lead to highly cytotoxic double strand breaks were quantified in a model system where double stranded DNA was sensitized to UV photons by labeling with 5-bromo-2'-deoxyuridine. The amount of breaks formed due to irradiation with several doses of 320nm photons was assayed by two independent methods: LC-MS and qPCR. A very good agreement between the relative damage measured by the two completely different analytical tools proves the applicability of qPCR for the quantitative analysis of SSBs. Our results suggest that the popularity of the hitherto underestimated though accurate and site-specific technique of real-time PCR may increase in future DNA damage studies. Copyright © 2016 Elsevier B.V. All rights reserved.
Okolie, Charles E
The genus Staphylococcus includes pathogenic and non-pathogenic facultative anaerobes. Due to the plethora of virulence factors encoded in its genome, the species Staphylococcus aureus is known to be the most pathogenic. S. aureus strains harboring genes encoding virulence and antibiotic resistance are of public health importance. In clinical samples, however, pathogenic S. aureus is often mixed with putatively less pathogenic coagulase-negative staphylococci (CoNS), both of which can harbor mecA, the genetic driver for staphylococcal methicillin-resistance. In this chapter, the detailed practical procedure for operating a real-time pentaplex PCR assay in blood cultures is described. The pentaplex real-time PCR assay simultaneously detects markers for the presence of bacteria (16S rRNA), coagulase-negative staphylococcus (cns), S. aureus (spa), Panton-Valentine leukocidin (pvl), and methicillin resistance (mecA).
Zocevic, Aleksandar; Vorimore, Fabien; Vicari, Nadia; Gasparini, Julien; Jacquin, Lisa; Sachse, Konrad; Magnino, Simone; Laroucau, Karine
Recent evidence of the occurrence of atypical Chlamydiaceae strains in pigeons, different from the established Chlamydiaceae, requires the development of a specific and rapid detection tool to investigate their prevalence and significance. Here is described a new real-time PCR assay that allows specific detection of atypical Chlamydiaceae from pigeons. The assay has been used to assess the dissemination of these strains in field samples collected from Parisian pigeon populations in 2009. The results suggest a limited dissemination compared to the usually higher prevalence of Chlamydia psittaci that is the main species associated with avian chlamydiosis. PMID:23516548
Huang, Meei-Li; Nguy, Long; Ferrenberg, James; Boeckh, Michael; Cent, Anne; Corey, Lawrence
Adenoviruses (AdV) have been associated with a wide variety of human disease and are increasingly recognized as viral pathogens that can cause significant morbidity and mortality in immunocompromised patients. Early detection of AdV DNA in plasma and sterile fluids has been shown to be useful for identifying patients at risk for invasive AdV disease. Due to the large number of existing Adv types, few real-time quantitative AdV PCR assays published effectively cover all AdV types. We designed a series of AdV PCR primers and probes and empirically multiplexed them into two separate real-time PCR assays to quantitatively detect all 49 serotypes of human AdV (Types 1-49) available from ATCC. We then subsequently multiplexed all the primers and probes into one reaction. The sensitivity of these assays was determined to be less than 10 copies per reaction (500 copies/ml plasma). In a retrospective evaluation we detected all 84 clinical AdV isolates isolated in cell culture from patients undergoing hematopoietic stem cell transplant (HSCT) between 1981 and 1987. Prospective analysis of 46 consecutive clinical samples submitted for adenovirus testing showed greater sensitivity and equal specificity of the AdV PCR than viral culture. This real time PCR assay allows rapid, sensitive and specific quantification of all currently defined adenoviruses into either two or one multiplex assay for clinical samples. PMID:18707838
Clothier, Kristin A; Jordan, Dianna M; Thompson, Curtis J; Kinyon, Joann M; Frana, Timothy S; Strait, Erin L
Mycoplasma bovis is an important bacterial pathogen in cattle, producing a variety of clinical diseases. The organism, which requires specialized culture conditions and extended incubation times to isolate and identify, is frequently associated with concurrent infection with other pathogens which can potentially be more easily identified. Real-time polymerase chain reaction (real-time PCR) is a valuable diagnostic technique that can rapidly identify infectious agents in clinical specimens. A real-time PCR assay was designed based on the uvrC gene to identify M. bovis in diagnostic samples. Using culture as the gold standard test, the assay performed well in a variety of diagnostic matrices. Initial validation testing was conducted on 122 milk samples (sensitivity: 88.9% [95% confidence interval (CI): 68.4-100%], specificity: 100%); 154 lung tissues (sensitivity: 89.0% [95% CI: 83.1-94.9%], specificity: 97.8% [95% CI: 93.5-100%]); 70 joint tissue/fluid specimens (sensitivity: 92.3% [95% CI: 82.1-100%], specificity: 95.5% [95% CI: 89.3-100%]); and 26 nasal swabs (sensitivity: 75.0% [95% CI: 45.0-100%], specificity: 83.3% [95% CI: 66.1-100%]). Low numbers of other sample matrices showed good agreement between results of culture and PCR. A review of clinical cases from 2009 revealed that, in general, PCR was used much more frequently than culture and provided useful diagnostic information in conjunction with clinical signs, signalment, and gross and histopathologic lesions. Diagnostic performance of the real-time PCR assay developed as a testing method indicates that it is a rapid, accurate assay that is adaptable to a variety of PCR platforms and can provide reliable results on an array of clinical samples.
Clinical validation of a novel diagnostic HIV-2 total nucleic acid qualitative assay using the Abbott m2000 platform: Implications for complementary HIV-2 nucleic acid testing for the CDC 4th generation HIV diagnostic testing algorithm.
Chang, Ming; Wong, Audrey J S; Raugi, Dana N; Smith, Robert A; Seilie, Annette M; Ortega, Jose P; Bogusz, Kyle M; Sall, Fatima; Ba, Selly; Seydi, Moussa; Gottlieb, Geoffrey S; Coombs, Robert W
The 2014 CDC 4th generation HIV screening algorithm includes an orthogonal immunoassay to confirm and discriminate HIV-1 and HIV-2 antibodies. Additional nucleic acid testing (NAT) is recommended to resolve indeterminate or undifferentiated HIV seroreactivity. HIV-2 NAT requires a second-line assay to detect HIV-2 total nucleic acid (TNA) in patients' blood cells, as a third of untreated patients have undetectable plasma HIV-2 RNA. To validate a qualitative HIV-2 TNA assay using peripheral blood mononuclear cells (PBMC) from HIV-2-infected Senegalese study participants. We evaluated the assay precision, sensitivity, specificity, and diagnostic performance of an HIV-2 TNA assay. Matched plasma and PBMC samples were collected from 25 HIV-1, 30 HIV-2, 8 HIV-1/-2 dual-seropositive and 25 HIV seronegative individuals. Diagnostic performance was evaluated by comparing the outcome of the TNA assay to the results obtained by the 4th generation HIV screening and confirmatory immunoassays. All PBMC from 30 HIV-2 seropositive participants tested positive for HIV-2 TNA including 23 patients with undetectable plasma RNA. Of the 30 matched plasma specimens, one was HIV non-reactive. Samples from 50 non-HIV-2 infected individuals were confirmed as non-reactive for HIV-2 Ab and negative for HIV-2 TNA. The agreement between HIV-2 TNA and the combined immunoassay results was 98.8% (79/80). Furthermore, HIV-2 TNA was detected in 7 of 8 PBMC specimens from HIV-1/HIV-2 dual-seropositive participants. Our TNA assay detected HIV-2 DNA/RNA in PBMC from serologically HIV-2 reactive, HIV indeterminate or HIV undifferentiated individuals with undetectable plasma RNA, and is suitable for confirming HIV-2 infection in the HIV testing algorithm. Copyright © 2016 Elsevier B.V. All rights reserved.
Yu, Zhongtang; Michel, Frederick C.; Hansen, Glenn; Wittum, Thomas; Morrison, Mark
We report here the development, validation, and use of three real-time PCR assays to quantify the abundance of the following three groups of tetracycline resistance genes: tet(A) and tet(C); tet(G); and tet genes encoding ribosomal protection proteins, including tet(M), tet(O), tetB(P), tet(Q), tet(S), tet(T), and tet(W). The assays were validated using known numbers of sample-derived tet gene templates added to microbiome DNA. These assays are both precise and accurate over at least 6 log tet gene copies. New tet gene variants were also identified from cloned tet amplicons as part of this study. The utility of these real-time PCR assays was demonstrated by quantifying the three tet gene groups present in bovine and swine manures, composts of swine manure, lagoons of hog house effluent, and samples from an Ekokan upflow biofilter system treating hog house effluent. The bovine manures were found to contain fewer copies of all three groups of tet genes than the swine manures. The composts of swine manures had substantially reduced tet gene abundance (up to 6 log), while lagoon storage or the upflow biofilter had little effect on tet gene abundance. These results suggest that the method of manure storage and treatment may have a substantial impact on the persistence and dissemination of tet genes in agricultural environments. These real-time PCR assays provide rapid, quantitative, cultivation-independent measurements of 10 major classes of tet genes, which should be useful for ecological studies of antibiotic resistance. PMID:16269727
Howard, Leigh M; Johnson, Monika; Gil, Ana I; Pekosz, Andrew; Griffin, Marie R; Edwards, Kathryn M; Lanata, Claudio F; Grijalva, Carlos G; Williams, John V
Influenza C virus (ICV) is associated with acute respiratory illness. Yet ICV remains under recognized, with most previous studies using only culture to identify cases. To develop a sensitive and specific real-time RT-PCR assay for ICV that allows for rapid and accurate detection in a clinical or research setting. Multiple ICV sequences obtained from GenBank were analyzed, including 141 hemagglutinin-esterase (HE), 106 matrix (M), and 97 nucleoprotein (NP) sequences. Primers and probes were designed based on conserved regions. Multiple primer-probe sets were tested against multiple ICV strains. The ICV M and NP genes offered the most conserved sequence regions. Primers and probes based on newer sequence data offered enhanced detection of ICV, especially for low titer specimens. An NP-targeted assay yielded the best performance and was capable of detecting 10-100 RNA copies per reaction. The NP assay detected multiple clinical isolates of ICV collected in a field epidemiology study conducted in Peru. We report a new real-time RT-PCR assay for ICV with high sensitivity and specificity. Copyright © 2017 Elsevier B.V. All rights reserved.
Shin, Hyewon; Kim, Minhwan; Yoon, Eunju; Kang, Gyoungwon; Kim, Seungyu; Song, Aelee; Kim, Jeongsoon
Staphylococcus aureus, the species most commonly associated with staphylococcal food poisoning, is one of the most prevalent causes of foodborne disease in Korea and other parts of the world, with much damage inflicted to the health of individuals and economic losses estimated at $120 million. To reduce food poisoning outbreaks by implementing prevention methods, rapid detection of S. aureus in foods is essential. Various types of detection methods for S. aureus are available. Although each method has advantages and disadvantages, high levels of sensitivity and specificity are key aspects of a robust detection method. Here, we describe a novel real-time isothermal target and probe amplification (iTPA) method that allows the rapid and simultaneous amplification of target DNA (the S. aureus nuc gene) and a fluorescence resonance energy transfer-based signal probe under isothermal conditions at 61 °C or detection of S. aureus in real time. The assay was able to specifically detect all 91 S. aureus strains tested without nonspecific detection of 51 non-S. aureus strains. The real-time iTPA assay detected S. aureus at an initial level of 10(1) CFU in overnight cultures of preenriched food samples (kiwi dressing, soybean milk, and custard cream). The advantage of this detection system is that it does not require a thermal cycler, reducing the cost of the real-time PCR and its footprint. Combined with a miniaturized fluorescence detector, this system can be developed into a simplified quantitative hand-held real-time device, which is often required. The iTPA assay was highly reliable and therefore may be used as a rapid and sensitive means of identifying S. aureus in foods.
Ikuta, Nilo; De Oliveira Solla Sobral, Fabiana; Lehmann, Fernanda Kieling Moreira; da Silveira, Proença Vinicius; de Carli, Silvia; Casanova, Yara Silva; Celmer, Álvaro José; Fonseca, André Salvador Kazantzi; Lunge, Vagner Ricardo
Avian pathogenic Escherichia coli (APEC) isolates are currently differentiated from nonpathogenic strains by classical PCR of virulence genes. This study improves the detection of the five main virulence genes used for APEC detection with the development of duplex and single Taqman real-time PCR to these targets. Primers and probes targeted to ompT, hlyF, iroN, iutA, and iss genes were designed and used in the implementation of single (iss) and duplex (hlyF/ompT and iroN/iutA) Taqman PCR assays. All five virulence genes of E coli strains were successfully detected by classical and Taqman real-time (single and duplex) PCR. A panel of 111 E coli isolates, obtained from avian samples collected in different Brazilian regions between 2010 and 2011, were further tested by both assays. Complete agreement was observed in the detection of four genes, ompT, hlyF, iron, iutA, but not for iss. This issue was addressed by combining the forward primer of the classical PCR to the new iss reverse primer and probe, resulting in complete agreement for all five genes. In total, 61 (55%) Brazilian E. coli isolates were detected as APEC, and the remaining 50 (45%) as avian fecal E. coli (AFEC). In conclusion, classical and Taqman real-time PCR presented exactly the same analytical performance for the differentiation of APEC and AFEC isolates. The developed real-time Taqman PCR assays could be used for the detection and differentiation of APEC isolates.
Huang, Huali; Cheng, Fang; Wang, Ruoan; Zhang, Dabing; Yang, Litao
Proper selection of endogenous reference genes and their real-time PCR assays is quite important in genetically modified organisms (GMOs) detection. To find a suitable endogenous reference gene and its real-time PCR assay for common wheat (Triticum aestivum L.) DNA content or copy number quantification, four previously reported wheat endogenous reference genes and their real-time PCR assays were comprehensively evaluated for the target gene sequence variation and their real-time PCR performance among 37 common wheat lines. Three SNPs were observed in the PKABA1 and ALMT1 genes, and these SNPs significantly decreased the efficiency of real-time PCR amplification. GeNorm analysis of the real-time PCR performance of each gene among common wheat lines showed that the Waxy-D1 assay had the lowest M values with the best stability among all tested lines. All results indicated that the Waxy-D1 gene and its real-time PCR assay were most suitable to be used as an endogenous reference gene for common wheat DNA content quantification. The validated Waxy-D1 gene assay will be useful in establishing accurate and creditable qualitative and quantitative PCR analysis of GM wheat. PMID:24098735
Huang, Huali; Cheng, Fang; Wang, Ruoan; Zhang, Dabing; Yang, Litao
Proper selection of endogenous reference genes and their real-time PCR assays is quite important in genetically modified organisms (GMOs) detection. To find a suitable endogenous reference gene and its real-time PCR assay for common wheat (Triticum aestivum L.) DNA content or copy number quantification, four previously reported wheat endogenous reference genes and their real-time PCR assays were comprehensively evaluated for the target gene sequence variation and their real-time PCR performance among 37 common wheat lines. Three SNPs were observed in the PKABA1 and ALMT1 genes, and these SNPs significantly decreased the efficiency of real-time PCR amplification. GeNorm analysis of the real-time PCR performance of each gene among common wheat lines showed that the Waxy-D1 assay had the lowest M values with the best stability among all tested lines. All results indicated that the Waxy-D1 gene and its real-time PCR assay were most suitable to be used as an endogenous reference gene for common wheat DNA content quantification. The validated Waxy-D1 gene assay will be useful in establishing accurate and creditable qualitative and quantitative PCR analysis of GM wheat.
Frank F. Roberto
Many techniques that are used to characterize and monitor microbial populations associated with sulfide mineral bioleaching require the cultivation of the organisms on solid or liquid media. Chemolithotrophic species, such as Acidithiobacillus ferrooxidans and Leptospirillum ferrooxidans, or thermophilic chemolithotrophs, such as Acidianus brierleyi and Sulfolobus solfataricus can grow quite slowly, requiring weeks to complete efforts to identify and quantify these microbes associated with bioleach samples. Real-time PCR (polymerase chain reaction) assays in which DNA targets are amplified in the presence of fluorescent oligonucleotide primers, allowing the monitoring and quantification of the amplification reactions as they progress, provide a means of rapidly detecting the presence of microbial species of interest, and their relative abundance in a sample. This presentation will describe the design and use of such assays to monitor acidophilic microbes in the environment and in bioleaching operations. These assays provide results within 2-3 hours, and can detect less than 100 individual microbial cells.
Lee, Rimi; Kim, Jihun; Kim, Sook Young; Jang, Seon Mi; Lee, Sun-Mi; Choi, In-Hong; Park, Seung Woo; Shin, Jeon-Soo; Yoo, Kyung-Hwa
Label-free cell-based assays have emerged as a promising means for high-throughput screening. Most label-free sensors are based on impedance measurements that reflect the passive electrical properties of cells. Here we introduce a capacitance-based assay that measures the dielectric constant (capacitance) of biological cells, and demonstrate the feasibility of analyzing endocytosis and screening chemotherapeutic agents with this assay. Endocytosis induces a change in the zeta potential, leading to a change in the dielectric constant which enables real-time endocytosis monitoring using the capacitance sensor. Additionally, since the dielectric constant is proportional to cell radius and cell volume, cell viability can be estimated from the change in capacitance. Therefore, the capacitance sensor array can also be used for cytotoxicity testing for large-scale chemotherapeutic screening.
Phister, Trevor G.; Mills, David A.
Traditional methods to detect the spoilage yeast Dekkera bruxellensis from wine involve lengthy enrichments. To overcome this difficulty, we developed a quantitative real-time PCR method to directly detect and enumerate D. bruxellensis in wine. Specific PCR primers to D. bruxellensis were designed to the 26S rRNA gene, and nontarget yeast and bacteria common to the winery environment were not amplified. The assay was linear over a range of cell concentrations (6 log units) and could detect as little as 1 cell per ml in wine. The addition of large amounts of nontarget yeasts did not impact the efficiency of the assay. This method will be helpful to identify possible routes of D. bruxellensis infection in winery environments. Moreover, the time involved in performing the assay (3 h) should enable winemakers to more quickly make wine processing decisions in order to reduce the threat of spoilage by D. bruxellensis. PMID:14660395
Phister, Trevor G; Mills, David A
Traditional methods to detect the spoilage yeast Dekkera bruxellensis from wine involve lengthy enrichments. To overcome this difficulty, we developed a quantitative real-time PCR method to directly detect and enumerate D. bruxellensis in wine. Specific PCR primers to D. bruxellensis were designed to the 26S rRNA gene, and nontarget yeast and bacteria common to the winery environment were not amplified. The assay was linear over a range of cell concentrations (6 log units) and could detect as little as 1 cell per ml in wine. The addition of large amounts of nontarget yeasts did not impact the efficiency of the assay. This method will be helpful to identify possible routes of D. bruxellensis infection in winery environments. Moreover, the time involved in performing the assay (3 h) should enable winemakers to more quickly make wine processing decisions in order to reduce the threat of spoilage by D. bruxellensis.
Hage, E; Mpamugo, O; Ohai, C; Sapkota, S; Swift, C; Wooldridge, D; Amar, C F L
The Listeria genus comprises 10 recognized species. Listeria monocytogenes causes listeriosis in humans and other animals primarily via contaminated food or animal feed. Listeria ivanovii causes listeriosis in animals and on rare occasions in humans. The identification of nonpathogenic species of Listeria in foods indicates that conditions exist that support the growth of pathogenic strains and is used to facilitate the implementation of control and prevention measures. This study shows the development and evaluation of a 5'exonuclease real-time PCR assay for the rapid identification of Listeria seeligeri, Listeria welshimeri, L. monocytogenes, L. ivanovii, Listeria grayi and Listeria innocua. The assay consists of two triplexes that were evaluated using 53 cultures of Gram-positive bacteria, including 49 Listeria spp. from human, animal, food or food-processing environments. The assay was rapid, specific and reproducible and could identify each of the six species from a mixture of strains. The developed assay proved to be a powerful means of rapidly identifying Listeria species and could be usefully implemented in busy specialist reference laboratories. The identification of species of Listeria from foods is important to monitor pathogenic strains and facilitates the implementation of control measures. This study shows the development and evaluation of a 5'exonuclease real-time PCR assay for the rapid identification of L. seeligeri, L. welshimeri, L. monocytogenes and L. ivanovii, L. grayi, L. innocua. The developed assay proved to be specific, rapid and reproducible and therefore could be implemented in busy specialist reference laboratories. © 2014 The Society for Applied Microbiology.
Rodríguez Lay, Licel de los Angeles; Montalvo Villalba, María Caridad; Sariego Frómeta, Susel; Bello Corredor, Marité; Mora Laguna, Elin; Kourí Cardellá, Vivian; Martínez Rodríguez, Pedro Ariel; Sánchez Wong, Meilin; Marrero, Bárbara
viral DNA levels in serum samples are a useful marker to monitor the disease progression and the treatment response in patients with chronic hepatitis B. Commercial kits for this purpose are available, but they are considerably expensive. to evaluate the analytical performance of a real-time polymerase chain reaction (RT-PCR) assay for Hepatitis B virus DNA quantification. specific primers to the gene C and TaqMan chemistry in a LightCycler 1.5 equipment was used. A standard curve was made and evaluated. Two hundred and seventy-two serum samples were used to assess the clinical and analytical specificity, the genotypic accuracy and specificity, the intra-assay and interassay coefficients of variation and the comparison with a commercial assay and with the qualitative PCR. the standard curve showed a strong linear correlation (r= -1) and low error values in the tested target DNA concentration. Analytical and clinical specificities were 100 %. Genotype accuracy and specificity showed that the differences between the results obtained by RT-PCR assay and those of the reference assay were less than 0.5 Log10. The 95% HBV DNA detection end-point assessed by Probit analysis was 16.41 IU/microL with a dynamic range of quantification of 10(8) IU/mL. Intra-assay and interassay coefficients of variation ranged from 0.16 to 1.45 % and 0.9 to 2.62 % respectively. The RT-PCR assay correlated well with those from a commercial assay (r= 0.964 and r2= 0.929) and with the HBV qualitative PCR, thus confirming its better sensitivity and advantages. the RT-PCR assay is well suited to monitoring HBV DNA levels showing to be sensitive, specific and reproducible. Its application in the clinical practice ensures a better diagnosis and management of patients with chronic hepatitis B in Cuba.
Matero, Pirjo; Pasanen, Tanja; Laukkanen, Riikka; Tissari, Päivi; Tarkka, Eveliina; Vaara, Martti; Skurnik, Mikael
A multiplex real-time polymerase chain reaction (PCR) assay was developed for the detection of Yersinia pestis and Yersinia pseudotuberculosis. The assay includes four primer pairs, two of which are specific for Y. pestis, one for Y. pestis and Y. pseudotuberculosis and one for bacteriophage lambda; the latter was used as an internal amplification control. The Y. pestis-specific target genes in the assay were ypo2088, a gene coding for a putative methyltransferase, and the pla gene coding for the plasminogen activator. In addition, the wzz gene was used as a target to specifically identify both Y. pestis and the closely related Y. pseudotuberculosis group. The primer and probe sets described for the different genes can be used either in single or in multiplex PCR assays because the individual probes were designed with different fluorochromes. The assays were found to be both sensitive and specific; the lower limit of the detection was 10-100 fg of extracted Y. pestis or Y. pseudotuberculosis total DNA. The sensitivity of the tetraplex assay was determined to be 1 cfu for the ypo2088 and pla probe labelled with FAM and JOE fluorescent dyes, respectively.
Miyagi, Yugo; Nomura, Hideyuki; Yamashita, Nobuyuki; Tanimoto, Hironori; Ito, Kiyoaki; Masaki, Naohiko; Mizokami, Masashi; Shibuya, Tsunefumi
Hepatitis C virus (HCV) RNA values measured with two real-time PCR methods (Cobas Ampliprep/Cobas TaqMan, CAP/CTM, and the Abbott real-time PCR test, ART) vary among patients with genotype 1. We investigated HCV RNA values measured by two real-time PCR assays during pegylated interferon plus ribavirin (PEG-IFN/RBV) therapy. We evaluated 185 cases of chronic hepatitis C patients, among which 97 patients received the PEG-IFN/RBV therapy. HCV RNA values of CAP/CTM for genotype 1 were significantly higher than those of ART (p < 0.05) The difference in HCV RNA values (CAP/CTM minus ART) of genotype 1 was significantly higher than those in genotype 2 (p < 0.0001). The positive rate (>0) of the difference of HCV RNA values in genotype 1 was 100 % (55/55), which was significantly higher than the 78.6 % (33/42) of genotype 2 (p < 0.001). There was no difference between TT and TG/GG genotype groups in terms of difference of HCV RNA values (CAP/CTM minus ART). After PEG-IFN/RBV therapy was administered, reduction of HCV measurements was observed from day 1 for both assays regardless of genotype. The HCV value of CAP/CTM during PEG-IFN/RBV therapy was consistently higher than the value of ART, although the difference in these two values gradually became smaller during the course of therapy, and eventually no significant difference was observed near the detection level. No correlation was observed between the sustained virological response (SVR) rate and the difference between the CAP/CTM HCV values and the ART HCV value before treatment.
Tatti, Kathleen M; Tondella, Maria Lucia
Bordetella pertussis causes an upper respiratory infection in infants, adolescents, and adults. Diagnosis of pertussis, a vaccine-preventable disease, can be difficult, but recent implementation of real-time PCR assays in laboratories has hastened the ability of clinicians to make an accurate diagnosis. In this paper we describe the method of nasopharyngeal specimen collection, extraction of DNA, and real-time PCR assays that will allow the detection and identification of Bordetella spp. in clinical specimens.
Lu, Xiaoyan; Whitaker, Brett; Sakthivel, Senthil Kumar K.; Kamili, Shifaq; Rose, Laura E.; Lowe, Luis; Mohareb, Emad; Elassal, Emad M.; Al-sanouri, Tarek; Haddadin, Aktham
A new human coronavirus (CoV), subsequently named Middle East respiratory syndrome (MERS)-CoV, was first reported in Saudi Arabia in September 2012. In response, we developed two real-time reverse transcription-PCR (rRT-PCR) assays targeting the MERS-CoV nucleocapsid (N) gene and evaluated these assays as a panel with a previously published assay targeting the region upstream of the MERS-CoV envelope gene (upE) for the detection and confirmation of MERS-CoV infection. All assays detected ≤10 copies/reaction of quantified RNA transcripts, with a linear dynamic range of 8 log units and 1.3 × 10−3 50% tissue culture infective doses (TCID50)/ml of cultured MERS-CoV per reaction. All assays performed comparably with respiratory, serum, and stool specimens spiked with cultured virus. No false-positive amplifications were obtained with other human coronaviruses or common respiratory viral pathogens or with 336 diverse clinical specimens from non-MERS-CoV cases; specimens from two confirmed MERS-CoV cases were positive with all assay signatures. In June 2012, the U.S. Food and Drug Administration authorized emergency use of the rRT-PCR assay panel as an in vitro diagnostic test for MERS-CoV. A kit consisting of the three assay signatures and a positive control was assembled and distributed to public health laboratories in the United States and internationally to support MERS-CoV surveillance and public health responses. PMID:24153118
Liu, Yan; Schanze, Kirk S
The fluorescence of the anionic, carboxylate-substituted poly(phenylene ethynylene) polymer PPECO2 is quenched very efficiently via the addition of 1 equiv of Cu(2+). Addition of pyrophosphate (PPi) into the weakly fluorescent solution of PPECO2 and Cu(2+) induces recovery of the polymer's fluorescence; the recovery occurs because PPi complexes with Cu(2+), effectively sequestering the ion so it cannot bind to the carboxylate groups of the polymer. A calibration curve was developed that relates the extent of fluorescence recovery to [PPi], making the PPECO2-Cu(2+) system a sensitive and selective turn-on sensor for PPi. Using the PPECO2-Cu(2+) system as the signal transducer, a real-time fluorescence turn-off assay for the enzyme alkaline phosphatase (ALP) using PPi as the substrate is developed. The assay operates with [PPi] in the micromolar range, and it offers a straightforward and rapid detection of ALP activity with the enzyme present in the nanomolar concentration range, operating either in an end point or real-time format. Kinetic and product inhibition parameters are derived by converting time-dependent fluorescence intensity into PPi (substrate) concentration, thus allowing calculation of the initial reaction rates (v(o)). Weak, nonspecific fluorescence responses are observed concomitant to addition of other proteins to the assay solution; however, the signal response to ALP is demonstrated to arise from the ALP catalyzed hydrolysis of PPi to phosphate (Pi).
Bae, Hi-Gung; Nitsche, Andreas; Teichmann, Anette; Biel, Stefan S; Niedrig, Matthias
Yellow fever virus quantitation is performed routinely by cultivation of virus containing samples using susceptible cells. Counting of the resulting plaques provides a marker for the number of infectious particles present in the sample. This assay usually takes up to 5 days before results are obtained and must be carried out under L2 or L3 laboratory conditions, depending on the yellow fever virus strain used. For clinical diagnosis of yellow fever virus infections the cell culture-based approach takes too long and is of limited practical relevance. Recently, due to its considerable sensitivity, PCR has become a promising method for virus detection. However, whilst PCR can detect virus-specific nucleic acids, it does not allow conclusions to be drawn regarding the infectious potential of the virus detected. Nonetheless, for diagnostic purposes, a rapid, specific and sensitive virus PCR is preferable. Therefore, two independent yellow fever virus-specific real-time PCR assays were established and compared the viral RNA loads to the results of a traditional plaque assay. The estimated ratio of yellow fever virus genomes to infectious particles was between 1000:1 and 5000:1; both approaches displayed a comparable precision of <45%. A significant correlation between genome number as determined by real-time PCR and the corresponding number of plaques in paired samples was found with a Pearson coefficient of correlation of r=0.88 (P<0.0001).
Cherkaoui, Abdessalam; Ceroni, Dimitri; Emonet, Stéphane; Lefevre, Yan; Schrenzel, Jacques
Kingella kingae is an emerging pathogen that is recognized as a causative agent of septic arthritis and osteomyelitis, primarily in infants and children. The bacterium is best detected by rapid inoculation in blood culture systems or by real-time PCR assays. Pathogenesis of the agent was linked recently to the production of a potent cytotoxin, known as RTX, which is toxic to a variety of human cell types. The locus encoding the RTX toxin is thought to be a putative virulence factor, and is, apparently, essential for inducing cytotoxic effects on respiratory epithelial, synovial and macrophage-like cells. Herein, we describe a novel real-time PCR assay that targets the RTX toxin gene and illustrate its use in two clinical cases. The assay exhibited a sensitivity of 30 c.f.u., which is 10-fold more sensitive than a previously published semi-nested broad-range 16S rRNA gene PCR, and showed no cross-reactivity with several related species and common osteoarticular pathogens.
Duressa, Dechassa; Rauscher, Gilda; Koike, Steven T; Mou, Beiquan; Hayes, Ryan J; Maruthachalam, Karunakaran; Subbarao, Krishna V; Klosterman, Steven J
Verticillium dahliae is a soilborne fungus that causes Verticillium wilt on multiple crops in central coastal California. Although spinach crops grown in this region for fresh and processing commercial production do not display Verticillium wilt symptoms, spinach seeds produced in the United States or Europe are commonly infected with V. dahliae. Planting of the infected seed increases the soil inoculum density and may introduce exotic strains that contribute to Verticillium wilt epidemics on lettuce and other crops grown in rotation with spinach. A sensitive, rapid, and reliable method for quantification of V. dahliae in spinach seed may help identify highly infected lots, curtail their planting, and minimize the spread of exotic strains via spinach seed. In this study, a quantitative real-time polymerase chain reaction (qPCR) assay was optimized and employed for detection and quantification of V. dahliae in spinach germplasm and 15 commercial spinach seed lots. The assay used a previously reported V. dahliae-specific primer pair (VertBt-F and VertBt-R) and an analytical mill for grinding tough spinach seed for DNA extraction. The assay enabled reliable quantification of V. dahliae in spinach seed, with a sensitivity limit of ≈1 infected seed per 100 (1.3% infection in a seed lot). The quantification was highly reproducible between replicate samples of a seed lot and in different real-time PCR instruments. When tested on commercial seed lots, a pathogen DNA content corresponding to a quantification cycle value of ≥31 corresponded with a percent seed infection of ≤1.3%. The assay is useful in qualitatively assessing seed lots for V. dahliae infection levels, and the results of the assay can be helpful to guide decisions on whether to apply seed treatments.
Background Bacterial load quantification is a critical component of bacterial community analysis, but a culture-independent method capable of detecting and quantifying diverse bacteria is needed. Based on our analysis of a diverse collection of 16 S rRNA gene sequences, we designed a broad-coverage quantitative real-time PCR (qPCR) assay—BactQuant—for quantifying 16 S rRNA gene copy number and estimating bacterial load. We further utilized in silico evaluation to complement laboratory-based qPCR characterization to validate BactQuant. Methods The aligned core set of 4,938 16 S rRNA gene sequences in the Greengenes database were analyzed for assay design. Cloned plasmid standards were generated and quantified using a qPCR-based approach. Coverage analysis was performed computationally using >670,000 sequences and further evaluated following the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines. Results A bacterial TaqMan® qPCR assay targeting a 466 bp region in V3-V4 was designed. Coverage analysis showed that 91% of the phyla, 96% of the genera, and >80% of the 89,537 species analyzed contained at least one perfect sequence match to the BactQuant assay. Of the 106 bacterial species evaluated, amplification efficiencies ranged from 81 to 120%, with r2-value of >0.99, including species with sequence mismatches. Inter- and intra-run coefficient of variance was <3% and <16% for Ct and copy number, respectively. Conclusions The BactQuant assay offers significantly broader coverage than a previously reported universal bacterial quantification assay BactQuant in vitro performance was better than the in silico predictions. PMID:22510143
Pauly, Diana; Worbs, Sylvia; Kirchner, Sebastian; Shatohina, Olena; Dorner, Martin B.; Dorner, Brigitte G.
Background In the context of a potential bioterrorist attack sensitive and fast detection of functionally active toxins such as ricin from complex matrices is necessary to be able to start timely countermeasures. One of the functional detection methods currently available for ricin is the endpoint cytotoxicity assay, which suffers from a number of technical deficits. Methodology/Findings This work describes a novel online cytotoxicity assay for the detection of active ricin and Ricinus communis agglutinin, that is based on a real-time cell electronic sensing system and impedance measurement. Characteristic growth parameters of Vero cells were monitored online and used as standardized viability control. Upon incubation with toxin the cell status and the cytotoxic effect were visualized using a characteristic cell index–time profile. For ricin, tested in concentrations of 0.06 ng/mL or above, a concentration-dependent decrease of cell index correlating with cytotoxicity was recorded between 3.5 h and 60 h. For ricin, sensitive detection was determined after 24 h, with an IC50 of 0.4 ng/mL (for agglutinin, an IC50 of 30 ng/mL was observed). Using functionally blocking antibodies, the specificity for ricin and agglutinin was shown. For detection from complex matrices, ricin was spiked into several food matrices, and an IC50 ranging from 5.6 to 200 ng/mL was observed. Additionally, the assay proved to be useful in detecting active ricin in environmental sample materials, as shown for organic fertilizer containing R. communis material. Conclusions/Significance The cell-electrode impedance measurement provides a sensitive online detection method for biologically active cytotoxins such as ricin. As the cell status is monitored online, the assay can be standardized more efficiently than previous approaches based on endpoint measurement. More importantly, the real-time cytotoxicity assay provides a fast and easy tool to detect active ricin in complex sample matrices. PMID
Ogawa, Eiichi; Furusyo, Norihiro; Murata, Masayuki; Hayashi, Takeo; Shimizu, Motohiro; Mukae, Haru; Toyoda, Kazuhiro; Hotta, Taeko; Uchiumi, Takeshi; Hayashi, Jun
Repeated measurement of the HCV RNA level is essential for properly monitoring treatment efficacy. The aim of this study was to determine the utility of two HCV real-time assays in the evaluation of the impact of hepatitis C virus (HCV) kinetics on the outcome of triple therapy with NS3/4A protease inhibitors (PIs), telaprevir or simeprevir. This study consisted of 171 Japanese patients infected with HCV genotype 1. All 3266 serum samples taken during and post treatment were tested with both the COBAS AmpliPrep/COBAS TaqMan (CAP/CTM) HCV Test v2.0 and the Abbott RealTime (ART) HCV Test. Of the 2597 samples undetectable (lower limit of detection [
Kwon, Seonhee; Jung, Bo Kyeung; Ko, Sun-Young; Lee, Chang Kyu
Background Quantitation of cytomegalovirus (CMV) DNA using real-time PCR has been utilized for monitoring CMV infection. However, the CMV antigenemia assay is still the 'gold standard' assay. There are only a few studies in Korea that compared the efficacy of use of real-time PCR for quantitation of CMV DNA in whole blood with the antigenemia assay, and most of these studies have been limited to transplant recipients. Method 479 whole blood samples from 79 patients, falling under different disease groups, were tested by real-time CMV DNA PCR using the Q-CMV real-time complete kit (Nanogen Advanced Diagnostic S.r.L., Italy) and CMV antigenemia assay (CINA Kit, ArgeneBiosoft, France), and the results were compared. Repeatedly tested patients were selected and their charts were reviewed for ganciclovir therapy. Results The concordance rate of the two assays was 86.4% (Cohen's kappa coefficient value=0.659). Quantitative correlation between the two assays was a moderate (r=0.5504, P<0.0001). Among 20 patients tested repeatedly with the two assays, 13 patients were transplant recipients and treated with ganciclovir. Before treatment, CMV was detected earlier by real-time CMV DNA PCR than the antigenemia assay, with a median difference of 8 days. After treatment, the antigenemia assay achieved negative results earlier than real-time CMV DNA PCR with a median difference of 10.5 days. Conclusions Q-CMV real-time complete kit is a useful tool for early detection of CMV infection in whole blood samples in transplant recipients. PMID:25553288
Garnier, Laurence; Gaudin, Jean-Christophe; Bensadoun, Paul; Rebillat, Isabelle; Morel, Yannick
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.
Pabbaraju, Kanti; Wong, Sallene; Wong, Anita A; Tellier, Raymond
Detection of all enteroviruses while excluding cross-detection of rhinoviruses is challenging because of sequence similarities in the commonly used conserved targets for molecular assays. In addition, simultaneous detection and differentiation of enteroviruses and parechoviruses would be beneficial because of a similar clinical picture presented by these viruses. A sensitive and specific real-time RT-PCR protocol that can address these clinical needs would be valuable to molecular diagnostic laboratories. Here we report a multiplex nucleic acid based assay using hydrolysis probes targeting the 5' non-translated region for the detection and differentiation of enteroviruses and parechoviruses without cross-detection of rhinoviruses. This assay has been shown to detect enteroviruses belonging to the different species in a variety of specimen types without detecting the different species of rhinoviruses. Laboratory validation shows the assay to be sensitive, specific, reproducible, easy to set up and uses generic cycling conditions. This assay can be implemented for diagnostic testing of patient samples in a high throughput fashion.
Liu, Zongxiao; Teng, Yong; Liu, Hong; Jiang, Yulin; Xie, Xiayang; Li, Huifang; Lv, Jiangqiang; Gao, Longying; He, Junqiang; Shi, Xiujie; Tian, Feiyan; Yang, Jingshun; Xie, Congxin
Spring viremia of carp virus (SVCV), infectious hematopoietic necrosis virus (IHNV) and viral hemorrhagic septicemia virus (VHSV) are three important fish rhabdoviruses, causing serious Office International des Epizooties (OIE) classified diseases in wild and farmed fish. Here, a new multiplex real-time quantitative RT-PCR (mqRT-PCR) assay was developed for simultaneous detection, identification and quantification of these three rhabdoviruses. The sets of primers and probes were targeted to conserved regions of glycoprotein (G) gene of SVCV, nucleoprotein (N) gene of IHNV and G gene of VHSV and used to amplify. The sensitivity, specificity and interference test of mqRT-PCR assay was analyzed. It was shown that the detection levels of 100 copies of SVCV, 220 copies of IHNV and 140 copies of VHSV were achieved, and there was no non-specific amplification and cross-reactivity using RNA of pike fry rhabdovirus (PFRV), infectious pancreatic necrosis virus (IPNV) and grass carp reovirus (GCRV). A total of 80 clinical fish samples were tested using the mqRT-PCR assay and the results were confirmed by antigen-capture ELISA and cell culture assay. This assay has the potential to be used for both research applications and diagnosis.
Briggs, R; Templeton, K; Fernando, I
Research suggests that some low-level virological rebound results occurring for no obvious clinical cause, in patients stable on antiretroviral therapy (ART), may be a consequence of the viral load assay used. We compared the relative frequency of clinically unexplained low-level virological rebound results when using the Roche HIV Taqman version-1 (CTM v1), the Roche HIV Taqman version-2 (CTM v2) and the Abbott RealTime (Abbott RT) assays in clinical practice. In all, 247 patients from our centre who had their viral loads measured by the three different assays over a period of 3 consecutive years (each assay used for a period of 1 year each) were included in the study. Low-level virological rebound was defined as <1000 copies/ml. Over similar time periods, there was significant discrepancy between the three assays when considering the proportion of clinically unexplained low-level virological rebound results in patients stable on ART: the CTM v2 assay produced the highest percentage (93%), CTM v1 much lower (65%) and Abbott RT even less (35%). There is further research required regarding what, if any, implications this has for patients who experience clinically unexplained low-level virological rebound on the more sensitive assays. © The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav.
Guillaud-Saumur, Thibaud; Nevez, Gilles; Bazire, Amélie; Virmaux, Michèle; Papon, Nicolas; Le Gal, Solène
We compared the RealCycler® PJIR kit (Progenie Molecular), available in Europe, to an in-house real-time PCR assay for the diagnosis of Pneumocystis jirovecii infections. Excellent agreement was found (concordance rate, 97.4%; Cohen's kappa, 0.918>0.8) showing that this commercial assay represents an alternative method for the diagnosis of P. jirovecii infections.
Wang, Hye-young; Kim, Sunghyun; Kim, Jungho; Park, Soon-Deok
Methicillin-resistant Staphylococcus aureus (MRSA) is the most prevalent cause of bloodstream infections (BSIs) and is recognized as a major nosocomial pathogen. This study aimed to evaluate a newly designed multiplex real-time PCR assay capable of the simultaneous detection of mecA, S. aureus, and coagulase-negative staphylococci (CoNS) in blood culture specimens. The Real-MRSA and Real-MRCoNS multiplex real-time PCR assays (M&D, Republic of Korea) use the TaqMan probes 16S rRNA for Staphylococcus spp., the nuc gene for S. aureus, and the mecA gene for methicillin resistance. The detection limit of the multiplex real-time PCR assay was 103 CFU/ml per PCR for each gene target. The multiplex real-time PCR assay was evaluated using 118 clinical isolates from various specimen types and a total of 350 positive blood cultures from a continuous monitoring blood culture system. The results obtained with the multiplex real-time PCR assay for the three targets were in agreement with those of conventional identification and susceptibility testing methods except for one organism. Of 350 positive bottle cultures, the sensitivities of the multiplex real-time PCR kit were 100% (166/166 cultures), 97.2% (35/36 cultures), and 99.2% (117/118 cultures) for the 16S rRNA, nuc, and mecA genes, respectively, and the specificities for all three targets were 100%. The Real-MRSA and Real-MRCoNS multiplex real-time PCR assays are very useful for the rapid accurate diagnosis of staphylococcal BSIs. In addition, the Real-MRSA and Real-MRCoNS multiplex real-time PCR assays could have an important impact on the choice of appropriate antimicrobial therapy, based on detection of the mecA gene. PMID:24648566
Wang, Hye-Young; Kim, Sunghyun; Kim, Jungho; Park, Soon-Deok; Uh, Young; Lee, Hyeyoung
Methicillin-resistant Staphylococcus aureus (MRSA) is the most prevalent cause of bloodstream infections (BSIs) and is recognized as a major nosocomial pathogen. This study aimed to evaluate a newly designed multiplex real-time PCR assay capable of the simultaneous detection of mecA, S. aureus, and coagulase-negative staphylococci (CoNS) in blood culture specimens. The Real-MRSA and Real-MRCoNS multiplex real-time PCR assays (M&D, Republic of Korea) use the TaqMan probes 16S rRNA for Staphylococcus spp., the nuc gene for S. aureus, and the mecA gene for methicillin resistance. The detection limit of the multiplex real-time PCR assay was 10(3) CFU/ml per PCR for each gene target. The multiplex real-time PCR assay was evaluated using 118 clinical isolates from various specimen types and a total of 350 positive blood cultures from a continuous monitoring blood culture system. The results obtained with the multiplex real-time PCR assay for the three targets were in agreement with those of conventional identification and susceptibility testing methods except for one organism. Of 350 positive bottle cultures, the sensitivities of the multiplex real-time PCR kit were 100% (166/166 cultures), 97.2% (35/36 cultures), and 99.2% (117/118 cultures) for the 16S rRNA, nuc, and mecA genes, respectively, and the specificities for all three targets were 100%. The Real-MRSA and Real-MRCoNS multiplex real-time PCR assays are very useful for the rapid accurate diagnosis of staphylococcal BSIs. In addition, the Real-MRSA and Real-MRCoNS multiplex real-time PCR assays could have an important impact on the choice of appropriate antimicrobial therapy, based on detection of the mecA gene.
Wright, Chelsea L; Hynes, Wayne L; White, Breanna T; Marshall, Mindy N; Gaff, Holly D; Gauthier, David T
Ixodes affinis Neumann (1899) and Ixodes scapularis Say (1821) are tick vectors of the etiologic agent of Lyme disease, Borrelia burgdorferi sensu stricto. Ixodes affinis and I. scapularis are morphologically very similar, and as they are sympatric in the mid- and south-Atlantic U.S. coastal states, their accurate identification is crucial to studies of disease and vector ecology in this area. This work describes a rapid, single-tube SYBR(®) Green-based real-time PCR assay for differentiation of I. affinis and I. scapularis at all life stages. The assay employs 2 pairs of species-specific primers directed against the internal transcribed spacer 2 (ITS2) region of the nuclear rRNA operon. Amplification products for these primer pairs differ in size and may be differentiated with a melt curve analysis. This tool is intended as a supplement to morphological methods for accurate identification of these ticks.
Dhami, Manpreet K.; Dsouza, Melissa; Waite, David W.; Anderson, Diane; Li, Dongmei
The brown marmorated stink bug, Halyomorpha halys (Hemiptera: Pentatomidae), is a gregarious crop pest that has rapidly spread across the world in the last two decades. It is an excellent hitchhiker species, especially as an over-wintering adult. During this period it is often associated with non-biological commodities such as shipping containers and machinery that travel long distances. Inadequate identification keys and similarity to common species has assisted its spread across Europe, while accurate identification from immature stages or eggs is not possible. We developed a real-time TaqMan PCR assay for the accurate and sensitive detection of the brown marmorated stink bug from all life stages. The assay performance against required diagnostic criterion and within a quarantine framework are described. PMID:26955631
Hamasuna, Ryoichi; Kawai, Shuichi; Ando, Yukiko; Ito, Kenji; Kurashima, Motoko; Nishimura, Hirohumi; Yamaguchi, Takamasa; Yoshimura, Makoto; Kobayashi, Tomoko; Muratani, Tetsuro; Matsumoto, Tetsuro
We evaluated performance of Abbott RealTime CT/NG assay (real-time PCR, Abbott Japan) for detect Chlamydia trachomatis and Neisseria gonorrhoeae by real-time PCR in 88 female patients with cervicitis symptoms seen at gynecological clinics and 100 male patients with urethritis symptoms seen at urological or dermatology clinics in Kitakyushu, Japan. Endocervical swab and first-voided urine (FVU) specimens were then collected from women and FVU specimens from men. Detection rates of C. trachomatis and N. gonorrhoeae by real-time PCR in the 3 types of specimens were compared to those by ProbeTec ET assay (ProbeTec, BD Diagnostic System). The overall positive concordance between real-time PCR and ProbTec were 97.1% (66/68) for C. trachomatis and 100% (33/33) for N. gonorrhoeae, C. trachomatis detection yielded 3 discordant results in endocervical specimens and 1 discordant result in male FVU by real-time PCR and ProbTec. Three of 4 reexamined using Aptime Combo 2 Assay (Fuji Rebio Inc.) were positive for C. trachomatis. Endocervical swab and FVU specimen results for C. trachomatis were discordant in 3 cases in real-time PCR and 4 in ProbeTec. Subjects with 2 or more positive endocervical awab results in female or male FVU specimens were assumed to be "true positive" for C. trachomatis. The sensitivities of real-time PCR for detecting C. trachomatis was 94.4% in endocervical swabs, 77.8% in female FVU and 97.4% in the male FVU. The sensitivities for real-time PCR for detectig N. gonorrhoeae was 100% in all 3 specimen types. Abbott RealTime CT/NG assay was useful for detecting C. trachomatis using endocervical swabs or male FVU specimens and for detecting N. gonorrhoeae using endocervical swabs and all FVU specimens.
Hamasuna, Ryoichi; Kawai, Shuichi; Ando, Yukiko; Ito, Kenji; Kurashima, Motoko; Nishimura, Hirohumi; Yamaguchi, Takamasa; Yoshimura, Makoto; Kobayashi, Tomoko; Muratani, Tetsuro; Matsumoto, Tetsuro
We evaluated performance of Abbott RealTime CT/NG assay (real-time PCR, Abbott Japan) for detect Chlamydia trachomatis and Neisseria gonorrhoeae by real-time PCR in 88 female patients with cervicitis symptoms seen at gynecological clinics and 100 male patients with urethritis symptoms seen at urological or dermatology clinics in Kitakyushu, Japan. Endocervical swab and first-voided urine (FVU) specimens were then collected from women and FVU specimens from men. Detection rates of C. trachomatis and N. gonorrhoeae by real-time PCR in the 3 types of specimens were compared to those by ProbeTec ET assay (ProbeTec, BD Diagnostic System). The overall positive concordance between real-time PCR and ProbTec were 97.1% (66/68) for C. trachomatis and 100% (33/33) for N. gonorrhoeae, C. trachomatis detection yielded 3 discordant results in endocervical specimens and 1 discordant result in male FVU by real-time PCR and ProbTec. Three of 4 reexamined using Aptime Combo 2 Assay (Fuji Rebio Inc.) were positive for C. trachomatis. Endocervical swab and FVU specimen results for C. trachomatis were discordant in 3 cases in real-time PCR and 4 in ProbeTec. Subjects with 2 or more positive endocervical awab results in female or male FVU specimens were assumed to be "true positive" for C. trachomatis. The sensitivities of real-time PCR for detecting C. trachomatis was 94.4% in endocervical swabs, 77.8% in female FVU and 97.4% in the male FVU. The sensitivities for real-time PCR for detecting N. gonorrhoeae was 100% in all 3 specimentypes. Abbott RealTime CT/NG assay was useful for detecting C. trachomatis using endocervical swabs or male FVU specimens and for detecting N. gonorrhoeae using endocervical swabs and all FVU specimens.
Bowers, H A; Tengs, T; Glasgow, H B; Burkholder, J M; Rublee, P A; Oldach, D W
Pfiesteria complex species are heterotrophic and mixotrophic dinoflagellates that have been recognized as harmful algal bloom species associated with adverse fish and human health effects along the East Coast of North America, particularly in its largest (Chesapeake Bay in Maryland) and second largest (Albermarle-Pamlico Sound in North Carolina) estuaries. In response to impacts on human health and the economy, monitoring programs to detect the organism have been implemented in affected areas. However, until recently, specific identification of the two toxic species known thus far, Pfiesteria piscicida and P. shumwayae (sp. nov.), required scanning electron microscopy (SEM). SEM is a labor-intensive process in which a small number of cells can be analyzed, posing limitations when the method is applied to environmental estuarine water samples. To overcome these problems, we developed a real-time PCR-based assay that permits rapid and specific identification of these organisms in culture and heterogeneous environmental water samples. Various factors likely to be encountered when assessing environmental samples were addressed, and assay specificity was validated through screening of a comprehensive panel of cultures, including the two recognized Pfiesteria species, morphologically similar species, and a wide range of other estuarine dinoflagellates. Assay sensitivity and sample stability were established for both unpreserved and fixative (acidic Lugol's solution)-preserved samples. The effects of background DNA on organism detection and enumeration were also explored, and based on these results, we conclude that the assay may be utilized to derive quantitative data. This real-time PCR-based method will be useful for many other applications, including adaptation for field-based technology.
Wang, Jianchang; Wang, Jinfeng; Liu, Libing; Yuan, Wanzhe
Porcine diseases associated with porcine circovirus 2 (PCV-2) infection have resulted in significant economic losses worldwide. A real-time recombinase polymerase amplification (RPA) assay was developed to detect PCV-2 using primers and an exo probe specific for the ORF2 gene. The reaction process can be completed in 20 min at 38 °C. The assay only detects PCV-2, as there was no cross-reaction with other pathogens important in pigs. Using the PCV-2 genomic DNA as template, the analytical sensitivity of the real-time RPA was 103 copies. The assay performance was evaluated by testing 38 field samples and compared with real-time PCR. The two assays demonstrated a 100% diagnostic agreement, and PCV-2 DNA was detected in 26 samples. The R(2) value of real-time RPA and real-time PCR was 0.954 by linear regression analysis. The real-time RPA assay provides an alternative tool for rapid, simple, and reliable detection of PCV-2, especially in remote and rural areas.
Kisner, H J
Many laboratorians have a limited perspective on what is involved in developing an instrument and bringing it to market. This article traces the product development process used by Abbott Diagnostics Division that resulted in Abbott being named the 1996 Concurrent Engineering Company of the Year for the design of the ARCHITECT.
... HUMAN SERVICES Food and Drug Administration 21 CFR Part 172 Abbott Laboratories; Filing of Food Additive Petition AGENCY: Food and Drug Administration, HHS. ACTION: Notice of petition. SUMMARY: The Food and Drug Administration (FDA) is announcing that Abbott Laboratories has filed a petition proposing that the food additive...
Evans, William N; Béland, Marie J
Few paediatric cardiologists know of Maude Abbott. Yet before Helen Taussig, no one contributed more to founding the specialty than Maude Abbott. She achieved international fame as the early 20th century expert on cardiac malformations. We summarise here her life and contributions, indicating how she is more than justified in being inducted to the Hall of Fame.
A new TaqMan real-time PCR assay was developed for the simultaneous quantitative detection of two seedborne maize pathogens in a single assay. Pantoea stewartii subsp. stewartii (Pnss) (syn. Erwinia stewartii) is the causal agent of Stewart's bacterial wilt and leaf blight of maize. Stewart's wilt i...
Balcázar, José Luis
Real-time PCR assays were developed for the enumeration of plasmid-mediated quinolone resistance (PMQR) determinants, such as the qnrA, qnrB, and qnrS genes, in different water samples and chicken feces. The results indicate that the developed assays are specific and sensitive for the quantification of qnr genes in complex samples. PMID:23275512
Tardif, Keith D; Schlaberg, Robert
Identification of Candida species by traditional methods can be time-consuming and have limited analytical sensitivity. We developed a multiplex real-time PCR assay for detection and differentiation of Candida species causing vulvovaginal candidiasis (VVC). Overall, this PCR assay is a powerful diagnostic tool offering superior accuracy, sensitivity, and specificity.
Savage, Harry M.
We assayed Zika virus–infected mosquitoes stored at room temperature for <30 days for live virus by using plaque assay and virus RNA by using real-time reverse transcription PCR. Viable virus was detected in samples stored <10 days, and virus RNA was detected in samples held for 30 days. PMID:28075325
Burkhalter, Kristen L; Savage, Harry M
We assayed Zika virus-infected mosquitoes stored at room temperature for <30 days for live virus by using plaque assay and virus RNA by using real-time reverse transcription PCR. Viable virus was detected in samples stored <10 days, and virus RNA was detected in samples held for 30 days.
O'Connell, Kevin P.; Bucher, Jennifer R.; Anderson, Patricia E.; Cao, Cheng J.; Khan, Akbar S.; Gostomski, Mark V.; Valdes, James J.
Bacteriophage MS2 is used in place of pathogenic viruses in a wide variety of studies that range from testing of compounds for disinfecting surfaces to studying environmental transport and fate of pathogenic viruses in groundwater. MS2 is also used as a pathogen simulant in the research, development, and testing (including open air tests) of methods, systems, and devices for the detection of pathogens in both the battlefield and homeland defense settings. PCR is often used as either an integral part of such detection systems or as a reference method to assess the sensitivity and specificity of microbial detection. To facilitate the detection of MS2 by PCR, we describe here a set of real-time fluorogenic reverse transcription-PCR assays. The sensitivity of the assays (performed with primer pairs and corresponding dye-labeled probes) ranged from 0.4 to 40 fg of MS2 genomic RNA (200 to 20,000 genome equivalents). We also demonstrate the usefulness of the primer pairs in assays without dye-labeled probe that included the DNA-binding dye SYBR green. None of the assays gave false-positive results when tested against 400 pg of several non-MS2 nucleic acid targets. PMID:16391081
Kuo, Hsiao-Che; Wang, Ting-Yu; Chen, Peng-Peng; Chen, Young-Mao; Chuang, Hui-Ching; Chen, Tzong-Yueh
Viral nervous necrosis caused by nervous necrosis virus (NNV) exacts a high mortality and results in huge economic losses in grouper aquaculture in Taiwan. The present study developed a real-time quantitative PCR (qPCR) method for NNV monitoring. The assay showed a strong linear correlation (r(2) = 0.99) between threshold cycle (C(T)) and RNA quantities, which allowed identification of infected groupers by the C(T) value and could be exploited to warn of NNV infection prior to an outbreak in grouper fish farms. Real-time qPCR also confirmed the copious content of NNV in grouper fin, similar to that in primary tissues; the result was verified by using in situ reverse transcription-PCR (RT-PCR). This indicated that grouper fin was a suitable sample for NNV detection, in a manner that could be relatively benign to the fish. The rapid spread of NNV infection to the entire population of affected farms was evident. The developed real-time qPCR method is rapid, highly sensitive, and applicable to routine high-throughput detection of large numbers of samples and has potential as a suitable tool for diagnostic, epidemiological, and genetic studies of grouper aquaculture.
Debski, Pawel R.; Gewartowski, Kamil; Bajer, Seweryn; Garstecki, Piotr
Quantitative Polymerase Chain Reaction (qPCR) is one of central techniques in molecular biology and important tool in medical diagnostics. While being a golden standard qPCR techniques depend on reference measurements and are susceptible to large errors caused by even small changes of reaction efficiency or conditions that are typically not marked by decreased precision. Digital PCR (dPCR) technologies should alleviate the need for calibration by providing absolute quantitation using binary (yes/no) signals from partitions provided that the basic assumption of amplification a single target molecule into a positive signal is met. Still, the access to digital techniques is limited because they require new instruments. We show an analog-digital method that can be executed on standard (real-time) qPCR devices. It benefits from real-time readout, providing calibration-free assessment. The method combines advantages of qPCR and dPCR and bypasses their drawbacks. The protocols provide for small simplified partitioning that can be fitted within standard well plate format. We demonstrate that with the use of synergistic assay design standard qPCR devices are capable of absolute quantitation when normal qPCR protocols fail to provide accurate estimates. We list practical recipes how to design assays for required parameters, and how to analyze signals to estimate concentration.
Debski, Pawel R.; Gewartowski, Kamil; Bajer, Seweryn; Garstecki, Piotr
Quantitative Polymerase Chain Reaction (qPCR) is one of central techniques in molecular biology and important tool in medical diagnostics. While being a golden standard qPCR techniques depend on reference measurements and are susceptible to large errors caused by even small changes of reaction efficiency or conditions that are typically not marked by decreased precision. Digital PCR (dPCR) technologies should alleviate the need for calibration by providing absolute quantitation using binary (yes/no) signals from partitions provided that the basic assumption of amplification a single target molecule into a positive signal is met. Still, the access to digital techniques is limited because they require new instruments. We show an analog-digital method that can be executed on standard (real-time) qPCR devices. It benefits from real-time readout, providing calibration-free assessment. The method combines advantages of qPCR and dPCR and bypasses their drawbacks. The protocols provide for small simplified partitioning that can be fitted within standard well plate format. We demonstrate that with the use of synergistic assay design standard qPCR devices are capable of absolute quantitation when normal qPCR protocols fail to provide accurate estimates. We list practical recipes how to design assays for required parameters, and how to analyze signals to estimate concentration. PMID:28327545
Svenstrup, Helle Friis; Jensen, Jørgen Skov; Björnelius, Eva; Lidbrink, Peter; Birkelund, Svend; Christiansen, Gunna
Mycoplasma genitalium is known to cause nonchlamydial, nongonococcal urethritis in men and to be associated with pelvic inflammatory disease in women. Specific and sensitive PCR methods are needed for diagnosis of this bacterium because it is very difficult to culture from patient samples. To determine the bacterial load in patients' specimens, a quantitative real-time LightCycler PCR was developed. The housekeeping gene gap encoding glyceraldehyde-3-phosphate dehydrogenase was chosen as the target gene. The assay could consistently detect five genome copies per reaction. To evaluate the PCR, we tested 246 selected urethral swab samples from men attending a clinic for sexually transmitted diseases. Eighty-two of the samples were found positive for M. genitalium by a conventional 16S rRNA gene PCR assay, whereas 164 samples were randomly chosen among those tested negative. Of the positive samples, 78 (95.1%) were found positive, whereas 6 (3.7%) of the negatives were found positive by the LightCycler assay. The patient samples were also tested with a quantitative TaqMan assay, and the bacterial load was compared to the LightCycler results. A good linear correlation between the LightCycler and the TaqMan assays was found with a correlation coefficient of 0.89 and a slope of 0.99. Significantly more M. genitalium-positive men had urethritis, discharge, and dysuria than had M. genitalium-negative men. The M. genitalium DNA load in samples from patients with urethritis was significantly higher than in samples from those without (61 and 2.9 copies/μl, respectively [P = 0.0005]). This assay may prove useful in the monitoring of treatment and for optimizing sample preparation methods. PMID:16000423
Tandon, Ravi; Cattori, Valentino; Willi, Barbara; Lutz, Hans; Hofmann-Lehmann, Regina
Endogenous retroviruses are integrated in the genome of most vertebrates. They represent footprints of ancient retroviral infection and are vertically transmitted from parents to their offspring. In the genome of all domestic cats, sequences closely related to exogenous FeLV known as endogenous feline leukemia virus (enFeLV), are present. enFeLV are incapable of giving rise to infectious virus particles. However, transcription and translation of enFeLV have been demonstrated in tissues of healthy cats and in feline cell lines. The presence of enFeLV-env has been shown in specific embryonic tissues and adult thymic cells. In addition, the enFeLV-env region recombines with FeLV subgroup A giving rise to an infectious FeLV-B virus. enFeLV envelope protein, FeLIX (FeLV infectivity X-essory protein) is also involved in mediating FeLV-T infection. In order to test the hypothesis that the enFeLV loads play a role in exogenous FeLV-A infection and pathogenesis, quantitative real-time PCR and RT-PCR assays were developed. An assay, specific to U3 region of all different subtypes of exogenous FeLV, was designed and applied to quantify exogenous FeLV proviral or viral load in cats, while three real-time PCR assays were designed to quantify U3 and env enFeLV loads (two within U3 amplifying different sequences; one within env). enFeLV loads were investigated in blood samples derived from Swiss privately owned domestic cats, specific pathogen-free (SPF) cats and European wildcats (Felis silvestris silvestris). Significant differences in enFeLV loads were observed between privately owned cats and SPF cats as well as among SPF cats originating from different catteries and among domestic cats of different breeds. When privately owned cats were compared, FeLV-infected cats had higher loads than uninfected cats. In addition, wildcats had higher enFeLV loads than domestic cats. In conclusion, the quantitative real-time PCR assays described herein are important prerequisites to
Nakamura, Alex A; Homem, Camila G; da Silva, Adriana M J; Meireles, Marcelo V
Three species and several genotypes of Cryptosporidium can infect the epithelial surface of the bursa of Fabricius, the respiratory tract, the proventriculus, the intestine, and the urinary tract in birds. There is reason to believe that gastric cryptosporidiosis in birds is caused by Cryptosporidium galli and Cryptosporidium avian genotype III, resulting in a chronic illness of the proventriculus that can lead to a debilitating and fatal clinical condition in birds of the orders Passeriformes and Psittaciformes. The objectives of the present study were to develop a duplex real-time polymerase chain reaction (PCR) that targets the 18S rRNA gene to simultaneously detect C. galli and Cryptosporidium avian genotype III DNA and to compare the duplex real-time PCR results to those of nested PCR targeting a partial fragment of the 18S rRNA gene, followed by sequencing of the amplified products (nPCR/S). A total of 1027 fecal samples were collected from birds of the orders Psittaciformes and Passeriformes originating either from captivity or the wild. Duplex real-time PCR results were positive in 580 (56.47%) and 21 (2.04%) samples, respectively, for C. galli and Cryptosporidium avian genotype III, whereas nPCR/S was positive in 28 (2.73%) and three (0.29%) samples, respectively, for C. galli and Cryptosporidium avian genotype III. Novel host birds were identified for both of the above gastric species, and it was also possible to identify Cryptosporidium baileyi and, for the first time in Brazil, Cryptosporidium avian genotype V. The duplex real-time PCR assay developed in the present study represents a sensitive and specific method for the detection of C. galli and Cryptosporidium avian genotype III in bird fecal samples. Moreover, this method may serve as an alternative to nPCR/S as a gold standard for the diagnosis of gastric cryptosporidiosis in birds.
van der Graaf-van Bloois, Linda; van Bergen, Marcel A P; van der Wal, Fimme J; de Boer, Albert G; Duim, Birgitta; Schmidt, Tracy; Wagenaar, Jaap A
Phenotypic differentiation between Campylobacter fetus (C. fetus) subspecies fetus and C. fetus subspecies venerealis is hampered by poor reliability and reproducibility of biochemical assays. AFLP (amplified fragment length polymorphism) and MLST (multilocus sequence typing) are the molecular standards for C. fetus subspecies identification, but these methods are laborious and expensive. Several PCR assays for C. fetus subspecies identification have been described, but a reliable comparison of these assays is lacking. The aim of this study was to evaluate the most practical and routinely implementable published PCR assays designed for C. fetus species and subspecies identification. The sensitivity and specificity of the assays were calculated by using an extensively characterized and diverse collection of C. fetus strains. AFLP and MLST identification were used as reference. Two PCR assays were able to identify C. fetus strains correctly at species level. The C. fetus species identification target, gene nahE, of one PCR assay was used to develop a real-time PCR assay with 100% sensitivity and 100% specificity, but the development of a subspecies venerealis specific real-time PCR (ISCfe1) failed due to sequence variation of the target insertion sequence and prevalence in other Campylobacter species. None of the published PCR assays was able to identify C. fetus strains correctly at subspecies level. Molecular analysis by AFLP or MLST is still recommended to identify C. fetus isolates at subspecies level.
Valero, Clara; de la Cruz-Villar, Laura; Zaragoza, Óscar; Buitrago, María José
The diagnosis of invasive fungal infections (IFIs) is usually based on the isolation of the fungus in culture and histopathological techniques. However, these methods have many limitations often delaying the definitive diagnosis. In recent years, molecular diagnostics methods have emerged as a suitable alternative for IFI diagnosis. When there is not a clear suspicion of the fungus involved in the IFI, panfungal real-time PCR assays have been used, allowing amplification of any fungal DNA. However, this approach requires subsequent amplicon sequencing to identify the fungal species involved, increasing response time. In this work, a new panfungal real-time PCR assay using the combination of an intercalating dye and sequence-specific probes was developed. After DNA amplification, a melting curve analysis was also performed. The technique was standardized by using 11 different fungal species and validated in 60 clinical samples from patients with proven and probable IFI. A melting curve database was constructed by collecting those melting curves obtained from fungal species included in the standardization assay. Results showed high reproducibility (coefficient of variation [CV] < 5%; r > 0.95) and specificity (100%). The overall sensitivity of the technique was 83.3%, with the group of fungi involved in the infection detected in 77.8% of the positive samples with IFIs covered by molecular beacon probes. Moreover, sequencing was avoided in 67.8% of these "probe-positive" results, enabling report of a positive result in 24 h. This technique is fast, sensitive, and specific and promises to be useful for improving early diagnosis of IFIs.
Collins, Catherine M; Kerr, Rose; McIntosh, Rebecca; Snow, Mike
Gyrodactylus salaris is a monogenean freshwater parasite that causes high mortality in wild Atlantic salmon, and a number of countries employ monitoring programmes for its presence. A TaqMan-MGB (minor groove binding) probe real-time multiplex assay targeting the internal transcribed spacer ribosomal DNA (ITS rDNA) was developed to simultaneously identify G. salaris/G. thymalli and 2 other commonly occurring Gyrodactylus species infecting salmonids in northern Europe: G. derjavinoides and G. truttae. In addition, a Gyrodactylus genus-level assay was developed to assess parasite DNA quality. The species-specific real-time PCR method correctly identified target species from a wide geographical range and from a number of salmonid hosts. It did not amplify G. lucii or G. teuchis. These species were successfully amplified using the Gyrodactylus genus real-time assay. The species-specific real-time assay proved to be significantly faster than the currently employed molecular screening method of ITS rDNA PCR amplification followed by restriction fragment length polymorphism analyses (RFLP). However, as with ITS RFLP, the real-time method did not distinguish between G. salaris and the non-pathogenic G. thymalli, its principle advantage being a significant reduction in time to achieve an initial diagnostic screen before the employment of more in-depth analyses for those specimens giving a positive G. salaris/G. thymalli real-time result.
Clancy, Eoin; Coughlan, Helena; Higgins, Owen; Boo, Teck Wee; Cormican, Martin; Barrett, Louise; Smith, Terry J; Reddington, Kate; Barry, Thomas
Three duplex molecular beacon based real-time Nucleic Acid Sequence Based Amplification (NASBA) assays have been designed and experimentally validated targeting RNA transcripts for the detection and identification of Haemophilus influenzae, Neisseria meningitidis and Streptococcus pneumoniae respectively. Each real-time NASBA diagnostics assay includes an endogenous non-competitive Internal Amplification Control (IAC) to amplify the splice variant 1 mRNA of the Homo sapiens TBP gene from human total RNA. All three duplex real-time NASBA diagnostics assays were determined to be 100% specific for the target species tested for. Also the Limits of Detection (LODs) for the H. influenzae, N. meningitidis and S. pneumoniae duplex real-time NASBA assays were 55.36, 0.99, and 57.24 Cell Equivalents (CE) respectively. These robust duplex real-time NASBA diagnostics assays have the potential to be used in a clinical setting for the rapid (<60min) specific detection and identification of the most prominent microorganisms associated with bacterial meningitis in humans.
van Beurden, S J; Voorbergen-Laarman, M A; Roozenburg, I; van Tellingen, J; Haenen, O L M; Engelsma, M Y
Anguillid herpesvirus 1 (AngHV1) causes a haemorrhagic disease with increased mortality in wild and farmed European eel, Anguilla anguilla (L.) and Japanese eel Anguilla japonica, Temminck & Schlegel). Detection of AngHV1 is currently based on virus isolation in cell culture, antibody-based typing assays or conventional PCR. We developed, optimized and concisely validated a diagnostic TaqMan probe based real-time PCR assay for the detection of AngHV1. The primers and probe target AngHV1 open reading frame 57, encoding the capsid protease and scaffold protein. Compared to conventional PCR, the developed real-time PCR is faster, less labour-intensive and has a reduced risk of cross-contamination. The real-time PCR assay was shown to be analytically sensitive and specific and has a high repeatability, efficiency and r(2) -value. The diagnostic performance of the assay was determined by testing 10% w/v organ suspensions and virus cultures from wild and farmed European eels from the Netherlands by conventional and real-time PCR. The developed real-time PCR assay is a useful tool for the rapid and sensitive detection of AngHV1 in 10% w/v organ suspensions from wild and farmed European eels.
Gunnell, Mark K; Lovelace, Charity D; Satterfield, Benjamin A; Moore, Emily A; O'Neill, Kim L; Robison, Richard A
Francisella tularensis is the aetiological agent of tularaemia, a zoonotic disease with worldwide prevalence. F. tularensis is a highly pathogenic organism and has been designated a category A biothreat agent by the Centers for Disease Control and Prevention. Tularaemia is endemic in much of the USA, Europe and parts of Asia. It is transmitted by numerous vectors and vehicles such as deer flies, ticks and rabbits. Currently, there are four recognized subspecies of F. tularensis: tularensis (type A), holarctica (type B), mediasiatica and novicida. Within the type A classification there are two subclassifications, type A.I and A.II, each with a specific geographical distribution across the USA. F. tularensis subsp. holartica (type B) is found in both the USA and Europe. Because of virulence differences among subtypes, it is important that health departments, hospitals and other government agencies are able to quickly identify each subtype. The purpose of this study was to develop a multiplex real-time PCR assay for the identification and discrimination of type A.I, type A.II, type B and novicida subspecies of F. tularensis. The assay was validated using 119 isolates of F. tularensis, three of its nearest neighbours and 14 other bacterial pathogens. This assay proved to be ~98 % successful at identifying the known subspecies of F. tularensis and could prove to be a useful tool in the characterization of this important pathogen.
Liu, Xin; Chen, Yu; Fierke, Carol A.
Ribonuclease P (RNase P) is an essential endonuclease that catalyzes the 5′ end maturation of precursor tRNA (pre-tRNA). Bacterial RNase P is an attractive potential antibacterial target because it is essential for cell survival and has a distinct subunit composition compared to the eukaryal counterparts. To accelerate both structure-function studies and discovery of inhibitors of RNase P, we developed the first real-time RNase P activity assay using fluorescence polarization/anisotropy (FP/FA) with a 5′ end fluorescein-labeled pre-tRNAAsp substrate. This FP/FA assay also detects binding of small molecules to pre-tRNA. Neomycin B and kanamycin B bind to pre-tRNAAsp with a Kd value that is comparable to their IC50 value for inhibition of RNase P, suggesting that binding of these antibiotics to the pre-tRNA substrate contributes to the inhibitory activity. This assay was optimized for high-throughput screening (HTS) to identify specific inhibitors of RNase P from a 2880 compound library. A natural product derivative, iriginol hexaacetate, was identified as a new inhibitor of Bacillus subtilis RNase P. The FP/FA methodology and inhibitors reported here will further our understanding of RNase P molecular recognition and facilitate discovery of antibacterial compounds that target RNase P. PMID:25249623
Hasanpour, Mojtaba; Najafi, Akram
Uropathogenic Escherichia coli (UPEC) is among major pathogens causing 80-90% of all episodes of urinary tract infections (UTIs). Recently, E. coli strains are divided into eight main phylogenetic groups including A, B1, B2, C, D, E, F, and clade I. This study was aimed to develop a rapid, sensitive, and specific multiplex real time PCR method capable of detecting phylogenetic groups of E. coli strains. This study was carried out on E. coli strains (isolated from the patient with UTI) in which the presence of all seven target genes had been confirmed in our previous phylogenetic study. An EvaGreen-based singleplex and multiplex real-time PCR with melting curve analysis was designed for simultaneous detection and differentiation of these genes. The primers were selected mainly based on the production of amplicons with melting temperatures (Tm) ranging from 82°C to 93°C and temperature difference of more than 1.5°C between each peak.The multiplex real-time PCR assays that have been developed in the present study were successful in detecting the eight main phylogenetic groups. Seven distinct melting peaks were discriminated, with Tm value of 93±0.8 for arpA, 89.2±0.1for chuA, 86.5±0.1 for yjaA, 82.3±0.2 for TspE4C2, 87.8±0.1for trpAgpC, 85.4±0.6 for arpAgpE genes, and 91±0.5 for the internal control. To our knowledge, this study is the first melting curve-based real-time PCR assay developed for simultaneous and discrete detection of these seven target genes. Our findings showed that this assay has the potential to be a rapid, reliable and cost-effective alternative for routine phylotyping of E. coli strains.
Viarouge, Cyril; Breard, Emmanuel; Zientara, Stephan; Vitour, Damien; Sailleau, Corinne
Epizootic haemorrhagic disease virus (EHDV) may cause severe clinical episodes in some species of deer and sometimes in cattle. Laboratory diagnosis provides a basis for the design and timely implementation of disease control measures. There are seven distinct EHDV serotypes, VP2 coding segment 2 being the target for serotype specificity. This paper reports the development and validation of eight duplex real-time RT-PCR assays to simultaneously amplify the EHDV target (S9 for the pan-EHDV real-time RT-PCR assay and S2 for the serotyping assays) and endogenous control gene Beta-actin. Analytical and diagnostic sensitivity and specificity, inter- and intra-assay variation and efficiency were evaluated for each assay. All were shown to be highly specific and sensitive. PMID:26161784
Wong, Samson S. Y.; Poon, Rosana W. S.; Chau, Sandy; Wong, Sally C. Y.; To, Kelvin K. W.; Cheng, Vincent C. C.; Fung, Kitty S. C.
Scabies remains the most prevalent, endemic, and neglected ectoparasitic infestation globally and can cause institutional outbreaks. The sensitivity of routine microscopy for demonstration of Sarcoptes scabiei mites or eggs in skin scrapings is only about 50%. Except for three studies using conventional or two-tube nested PCR on a small number of cases, no systematic study has been performed to improve the laboratory diagnosis of this important infection. We developed a conventional and a real-time quantitative PCR (qPCR) assay based on the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene of S. scabiei. The cox1 gene is relatively well conserved, with its sequence having no high levels of similarity to the sequences of other human skin mites, pathogenic zoonotic mites, or common house dust mite species. This mitochondrial gene is also present in large quantities in arthropod cells, potentially improving the sensitivity of a PCR-based assay. In our study, both assays were specific and were more sensitive than microscopy in diagnosing scabies, with positive and negative predictive values of 100%. The S. scabiei DNA copy number in the microscopy-positive specimens was significantly higher than that in the microscopy-negative specimens (median S. scabiei DNA copy number, 3.604 versus 2.457 log10 copies per reaction; P = 0.0213). In the patient with crusted scabies, the qPCR assay performed on lesional skin swabs instead of scrapings revealed that the parasite DNA load took about 2 weeks to become negative after treatment. The utility of using lesional skin swabs as an alternative sample for diagnosis of scabies by PCR should be further evaluated. PMID:25903566
Wong, Samson S Y; Poon, Rosana W S; Chau, Sandy; Wong, Sally C Y; To, Kelvin K W; Cheng, Vincent C C; Fung, Kitty S C; Yuen, K Y
Scabies remains the most prevalent, endemic, and neglected ectoparasitic infestation globally and can cause institutional outbreaks. The sensitivity of routine microscopy for demonstration of Sarcoptes scabiei mites or eggs in skin scrapings is only about 50%. Except for three studies using conventional or two-tube nested PCR on a small number of cases, no systematic study has been performed to improve the laboratory diagnosis of this important infection. We developed a conventional and a real-time quantitative PCR (qPCR) assay based on the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene of S. scabiei. The cox1 gene is relatively well conserved, with its sequence having no high levels of similarity to the sequences of other human skin mites, pathogenic zoonotic mites, or common house dust mite species. This mitochondrial gene is also present in large quantities in arthropod cells, potentially improving the sensitivity of a PCR-based assay. In our study, both assays were specific and were more sensitive than microscopy in diagnosing scabies, with positive and negative predictive values of 100%. The S. scabiei DNA copy number in the microscopy-positive specimens was significantly higher than that in the microscopy-negative specimens (median S. scabiei DNA copy number, 3.604 versus 2.457 log10 copies per reaction; P = 0.0213). In the patient with crusted scabies, the qPCR assay performed on lesional skin swabs instead of scrapings revealed that the parasite DNA load took about 2 weeks to become negative after treatment. The utility of using lesional skin swabs as an alternative sample for diagnosis of scabies by PCR should be further evaluated. Copyright © 2015, American Society for Microbiology. All Rights Reserved.
Jalal, Hamid; Stephen, Hannah; Alexander, Sarah; Carne, Christopher; Sonnex, Christopher
We have developed and validated a nested real-time PCR (NRT-PCR) for the genotyping of Chlamydia trachomatis and used it specifically for the typing of either eight genovars from D to K or three genovars of lymphogranuloma venereum (LGV). The 11 probes used in the NRT-PCR correctly identified the DNA from D to K and LGV reference strains and did not cross-react with the DNA from 26 strains representing the bacterial pathogens and commensals of the oropharynx, genital tract, and rectum. The NRT-PCR had a 95% probability of detection at four genome copies (confidence interval, three to six copies) of C. trachomatis per reaction. One hundred cervical and urethral swab specimens containing C. trachomatis DNA from 63 women and 37 men were used to validate the method. The results from the NRT-PCR and the DNA sequencing of amplicons generated from the omp1 gene showed 100% correlation for these samples. The assay also identified the LGV-II genotype in 24 of 48 rectal swab specimens containing C. trachomatis DNA that were obtained from men having sex with men. The Sexually Transmitted Bacteria Reference Laboratory, London, independently confirmed these results using group-specific LGV real-time PCR and restriction fragment length polymorphism analysis. Compared with the NRT-PCR, non-NRT-PCR was found to be less sensitive: it typed C. trachomatis DNA in only 80% of the genital samples and 90% of the rectal swab samples. This is the first successful demonstration of the use of real-time PCR for the genotype-specific typing of C. trachomatis strains that cause sexually transmitted diseases.
Park, Hee Kuk; Lee, Hee Joong; Lee, Hee Jung; Kim, Wonyong
Streptococcus pneumoniae is the main etiologic agent of pneumonia worldwide. Because the members of the viridans group streptococci share a high degree of DNA sequence homologies, phenotypic and genotypic discriminations of S. pneumoniae from the viridans group are difficult. A quantitative real-time PCR assay targeting the capsular polysaccharide biosynthesis gene (cpsA) was developed as a species-specific detection tool for S. pneumoniae. The specificity was evaluated using genomic DNAs extracted from 135 oral cocci strains. Twenty-seven S. pneumoniae strains tested positive, whereas 108 other strains including Streptococcus pseudopneumoniae, Streptococcus mitis, and Streptococcus oralis did not show a specific signal. The linear regression of standard curves indicated high correlations between the log numbers of S. pneumoniae cells and the C(T) values (R(2)=0.99). The minimal limit of detection was 32 fg of purified genomic DNA, equivalent to 14 genomes of S. pneumoniae. This new real-time PCR method may be very useful as a rapid and specific tool for detecting and quantifying S. pneumoniae.
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
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. Copyright © 2016 Elsevier B.V. All rights reserved.
Irenge, Léonid M; Durant, Jean-François; Tomaso, Herbert; Pilo, Paola; Olsen, Jaran S; Ramisse, Vincent; Mahillon, Jacques; Gala, Jean-Luc
A real-time polymerase chain reaction (PCR) assay was developed for rapid identification of Bacillus anthracis in environmental samples. These samples often harbor Bacillus cereus bacteria closely related to B. anthracis, which may hinder its specific identification by resulting in false positive signals. The assay consists of two duplex real-time PCR: the first PCR allows amplification of a sequence specific of the B. cereus group (B. anthracis, B. cereus, Bacillus thuringiensis, Bacillus weihenstephanensis, Bacillus pseudomycoides, and Bacillus mycoides) within the phosphoenolpyruvate/sugar phosphotransferase system I gene and a B. anthracis specific single nucleotide polymorphism within the adenylosuccinate synthetase gene. The second real-time PCR assay targets the lethal factor gene from virulence plasmid pXO1 and the capsule synthesis gene from virulence plasmid pXO2. Specificity of the assay is enhanced by the use of minor groove binding probes and/or locked nucleic acids probes. The assay was validated on 304 bacterial strains including 37 B. anthracis, 67 B. cereus group, 54 strains of non-cereus group Bacillus, and 146 Gram-positive and Gram-negative bacteria strains. The assay was performed on various environmental samples spiked with B. anthracis or B. cereus spores. The assay allowed an accurate identification of B. anthracis in environmental samples. This study provides a rapid and reliable method for improving rapid identification of B. anthracis in field operational conditions.
Neumann, A P; Dunham, S M; Rehberger, T G; Siragusa, G R
Clostridium septicum is a spore-forming anaerobe frequently implicated in cases of gangrenous dermatitis (GD) and other spontaneously occurring myonecrotic infections of poultry. Although C. septicum is readily cultured from diseased tissues it can be difficult to enumerate due to its tendency to swarm over the surface of agar plates. In this study a quantitative real-time PCR assay was developed in order to more accurately measure the levels of C. septicum in healthy as well as GD associated poultry samples. The assay was specifically designed to target the C. septicum alpha toxin gene, csa, which is, to our knowledge, carried by all strains of C. septicum and has been shown to be essential for virulence. Genomic DNAs from a diverse collection of bacterial species, including closely related Clostridium chauvoei, Clostridium carnis, Clostridium tertium as well as several strains of Clostridium perfringens, all failed to produce a positive reaction. An approximate reproducible limit of detection in spiked extracts of at least 10(3) cfu/g of C. septicum was observed for a variety of different sample types. C. septicum levels in broiler chicken field samples estimated from the results of qPCR were statistically correlated to culture based enumerations obtained from those same tissues.
Angelini, Claudia; Carfora, Maria Francesca; Carriero, Maria Vincenza; Natalini, Roberto
Experiments of cell migration and chemotaxis assays have been classically performed in the so-called Boyden Chambers. A recent technology, xCELLigence Real Time Cell Analysis, is now allowing to monitor the cell migration in real time. This technology measures impedance changes caused by the gradual increase of electrode surface occupation by cells during the course of time and provide a Cell Index which is proportional to cellular morphology, spreading, ruffling and adhesion quality as well as cell number. In this paper we propose a macroscopic mathematical model, based on advection-reaction-diffusion partial differential equations, describing the cell migration assay using the real-time technology. We carried out numerical simulations to compare simulated model dynamics with data of observed biological experiments on three different cell lines and in two experimental settings: absence of chemotactic signals (basal migration) and presence of a chemoattractant. Overall we conclude that our minimal mathematical model is able to describe the phenomenon in the real time scale and numerical results show a good agreement with the experimental evidences. PMID:27680883
Di Costanzo, Ezio; Ingangi, Vincenzo; Angelini, Claudia; Carfora, Maria Francesca; Carriero, Maria Vincenza; Natalini, Roberto
Experiments of cell migration and chemotaxis assays have been classically performed in the so-called Boyden Chambers. A recent technology, xCELLigence Real Time Cell Analysis, is now allowing to monitor the cell migration in real time. This technology measures impedance changes caused by the gradual increase of electrode surface occupation by cells during the course of time and provide a Cell Index which is proportional to cellular morphology, spreading, ruffling and adhesion quality as well as cell number. In this paper we propose a macroscopic mathematical model, based on advection-reaction-diffusion partial differential equations, describing the cell migration assay using the real-time technology. We carried out numerical simulations to compare simulated model dynamics with data of observed biological experiments on three different cell lines and in two experimental settings: absence of chemotactic signals (basal migration) and presence of a chemoattractant. Overall we conclude that our minimal mathematical model is able to describe the phenomenon in the real time scale and numerical results show a good agreement with the experimental evidences.
Zhang, An Ni; Mao, Yanping; Zhang, Tong
We designed novel quantitative real-time polymerase chain reaction (qPCR) primers for the polyphosphate kinase 1 (ppk1) gene, targeting eight individual “Candidatus Accumulibacter” (referred to as Accumulibacter) clades. An evaluation of primer sets was conducted regarding the coverage, specificity, and PCR efficiency. (i) All primer sets were designed to cover all available sequences of the target clade. (ii) The phylogenetic analysis of the sequences retrieved from the qPCR products by each primer set demonstrated a high level of specificity. (iii) All calibration curves presented high PCR efficiencies in the range of 85-112% (R2 = 0.962-0.998). In addition, the possible interference of non-target amplicons was individually examined using the qPCR assay for 13 Accumulibacter clades, which were either undetected or showed negligible detection. With the primers designed by other research groups, a highly selective and sensitive qPCR-based method was developed to quantify all Accumulibacter clades, with the exception of Clade IE, in one assay, which enables more comprehensive insights into the community dynamics. The applicability to environmental samples was demonstrated by profiling the Accumulibacter clades in activated sludge samples of nine full-scale wastewater treatment plants.
Zhang, An Ni; Mao, Yanping; Zhang, Tong
We designed novel quantitative real-time polymerase chain reaction (qPCR) primers for the polyphosphate kinase 1 (ppk1) gene, targeting eight individual “Candidatus Accumulibacter” (referred to as Accumulibacter) clades. An evaluation of primer sets was conducted regarding the coverage, specificity, and PCR efficiency. (i) All primer sets were designed to cover all available sequences of the target clade. (ii) The phylogenetic analysis of the sequences retrieved from the qPCR products by each primer set demonstrated a high level of specificity. (iii) All calibration curves presented high PCR efficiencies in the range of 85–112% (R2 = 0.962–0.998). In addition, the possible interference of non-target amplicons was individually examined using the qPCR assay for 13 Accumulibacter clades, which were either undetected or showed negligible detection. With the primers designed by other research groups, a highly selective and sensitive qPCR-based method was developed to quantify all Accumulibacter clades, with the exception of Clade IE, in one assay, which enables more comprehensive insights into the community dynamics. The applicability to environmental samples was demonstrated by profiling the Accumulibacter clades in activated sludge samples of nine full-scale wastewater treatment plants. PMID:27142574
Bowers, Holly A.; Tomas, Carmelo; Tengs, Torstein; Kempton, Jason W.; Lewitus, Alan J.; Oldach, David W.
Species within the class Raphidophyceae were associated with fish kill events in Japanese, European, Canadian, and U.S. coastal waters. Fish mortality was attributable to gill damage with exposure to reactive oxygen species (peroxide, superoxide, and hydroxide radicals), neurotoxins, physical clogging, and hemolytic substances. Morphological identification of these organisms in environmental water samples is difficult, particularly when fixatives are used. Because of this difficulty and the continued global emergence of these species in coastal estuarine waters, we initiated the development and validation of a suite of real-time polymerase chain reaction (PCR) assays. Sequencing was used to generate complete data sets for nuclear encoded small-subunit ribosomal RNA (SSU rRNA; 18S); internal transcribed spacers 1 and 2, 5.8S; and plastid encoded SSU rRNA (16S) for confirmed raphidophyte cultures from various geographic locations. Sequences for several Chattonella species (C. antiqua, C. marina, C. ovata, C. subsalsa, and C. verruculosa), Heterosigma akashiwo, and Fibrocapsa japonica were generated and used to design rapid and specific PCR assays for several species including C. verruculosa Hara et Chihara, C. subsalsa Biecheler, the complex comprised of C. marina Hara et Chihara, C. antiqua Ono and C. ovata, H. akashiwo Ono, and F. japonica Toriumi et Takano using appropriate loci. With this comprehensive data set, we were also able to perform phylogenetic analyses to determine the relationship between these species. PMID:20411032
Korchinski, Katie L; Land, Adrian D; Craft, David L; Brzezinski, Jennifer L
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.
Kesmen, Zulal; Yetiman, Ahmet E; Sahin, Fikrettin; Yetim, Hasan
In this study, TaqMan-based real-time Polymerase Chain Reaction (PCR) techniques were developed for the detection of chicken and turkey meat in raw and heat-treated meat mixtures. Primers and TaqMan probe sets were designed to amplify 86 bp and 136 bp fragments for the chicken and turkey species, respectively, on the mitochondrial NADH dehydrogenase subunit 2 gene. In the results, it was possible to detect each species at the level of 0.1 pg template DNA with the TaqMan probe technique without any cross-reactivity with nontarget species (bovine, ovine, donkey, pork, and horse) while the detection level was 1 pg template DNA using conventional PCR. The TaqMan probe assays used in this study allowed the detection of as little as 0.001% level of both species in the experimental meat mixtures, prepared by mixing chicken and turkey meat with beef at different levels (0.001% to 10%). In conclusion, TaqMan probe assays developed in this research are promising tools in the specific identification and sensitive quantification of meat species even in the case of heat-treated meat products, and suitable for a rapid, automated, and routine analysis.
Halm, Anna; Wagner, Martin; Köfer, Josef; Hein, Ingeborg
A real-time PCR assay based on the 16S rRNA gene sequence was designed for differentiation of blackleg-causing Clostridium chauvoei and Clostridium septicum, a phylogenetically closely related bacterium responsible for malignant edema. In order to exclude false-negative results, an internal amplification control was included in the assay. A set of three probes, one specific for C. chauvoei, one specific for C. septicum, and one specific for both species, permitted unequivocal detection of C. chauvoei in tests of 32 Clostridium sp. strains and 10 non-Clostridium strains. The assay proved to be sensitive, detecting one genome of C. chauvoei or C. septicum per PCR and 1.79 × 103 C. chauvoei cells/g artificially contaminated muscle tissue. In tests of 11 clinical specimens, the real-time PCR assay yielded the same results as an established conventional PCR method. PMID:20129968
Halm, Anna; Wagner, Martin; Köfer, Josef; Hein, Ingeborg
A real-time PCR assay based on the 16S rRNA gene sequence was designed for differentiation of blackleg-causing Clostridium chauvoei and Clostridium septicum, a phylogenetically closely related bacterium responsible for malignant edema. In order to exclude false-negative results, an internal amplification control was included in the assay. A set of three probes, one specific for C. chauvoei, one specific for C. septicum, and one specific for both species, permitted unequivocal detection of C. chauvoei in tests of 32 Clostridium sp. strains and 10 non-Clostridium strains. The assay proved to be sensitive, detecting one genome of C. chauvoei or C. septicum per PCR and 1.79 x 10(3) C. chauvoei cells/g artificially contaminated muscle tissue. In tests of 11 clinical specimens, the real-time PCR assay yielded the same results as an established conventional PCR method.
Aldewachi, Hasan Saad; Woodroofe, Nicola; Turega, Simon; Gardiner, Philip H E
Dipeptidyl peptidase IV (DPP-IV also referred to as CD-26) is a serine protease enzyme with remarkable diagnostic and prognostic value in a variety of health and disease conditions. Herein, we describe a simple and real-time colorimetric assay for DPP-IV/CD-26 activity based on the aggregation of gold nanoparticles (AuNPs) functionalized with the peptide substrates: Gly-Pro-Asp-Cys (GPDC) or Val-Pro-ethylene diamine-Asp-Cys (VP-ED-DC). Cleavage of the substrates by DPP-IV resulted in aggregation of the AuNPs with accompanying color change in the solution from red to blue that was monitored using either a UV-visible spectrophotometer or by the naked eye. Factors, such as time course of the reaction, stability of the functionalized AuNPs and the structure of the substrate that influence the cleavage reaction in solution were investigated. The effects of potential interference from serum proteins (lysozyme, thrombin and trypsin) on the analytical response were negligible. The detection limits when GPDC or VP-EN-DC functionalized AuNPs were used for DPP-IV assay were 1.2U/L and 1.5U/L, respectively. The VP-EN-DC method was preferred for the quantitative determination of DPP-IV activity in serum because of its wide linear range 0-30U/L compared to 0-12U/L for the GPDC assay. Recoveries from serum samples spiked with DPP-IV activity, between 5 and 25U/L, and using the VP-EN-DC modified AuNPs method ranged between 83.6% and 114.9%. The two colorimetric biosensors described here are superior to other conventional methods because of their simplicity, stability, selectivity and reliability. Copyright © 2017 Elsevier B.V. All rights reserved.
Jazeron, Jean-François; Barbe, Coralie; Frobert, Emilie; Renois, Fanny; Talmud, Déborah; Brixi-Benmansour, Hedia; Brodard, Véronique; Andréoletti, Laurent; Diebold, Marie-Danièle
Herpes simplex virus 1 (HSV-1) esophagitis diagnosis is routinely based on the endoscopic findings confirmed by histopathological examination of the esophagitis lesions. Virological diagnosis is not systematically performed and restricted to viral culture or to qualitative PCR assay from esophagitis biopsy specimens. The aim of this study was to assess the interest of quantitative real-time PCR assay in HSV-1 esophagitis diagnosis by comparing the results obtained to those of histological examination associated with immunohistochemical staining, which is considered the “gold standard.” From 53 esophagitis biopsy specimens, the PCR assay detected HSV-1 in 18 of 19 histologically proven to have herpetic esophagitis and in 9 of 34 that had esophagitis related to other causes, demonstrating sensitivity, specificity, positive predictive value, and negative predictive value of 94.7%, 73%, 66.7%, and 96%, respectively. Interestingly, HSV-1 was not detected in 16 specimens without the histological aspect of esophagitis. The viral loads normalized per μg of total extracted DNA in each biopsy specimen detected positive by HSV PCR were then compared and appeared to be significantly higher in histopathologically positive herpetic esophagitis (median = 2.9 × 106 ± 1.1 × 108) than in histopathologically negative herpetic esophagitis (median = 3.1 × 103 ± 6.2 × 103) (P = 0.0009). Moreover, a receiver operating characteristics analysis revealed that a viral load threshold greater than 2.5 × 104 copies would allow an HSV-1 esophagitis diagnosis with a sensitivity and specificity of 83.3% and 100%, respectively. In conclusion, this work demonstrated that HSV quantitative PCR results for paraffin-embedded esophageal tissue was well correlated to histopathological findings for an HSV-1 esophagitis diagnosis and could be diagnostic through viral load assessment when histopathological results are missing or uncertain. PMID:22170921
Wang, Deguo; Wang, Yongzhen; Xiao, Fugang; Guo, Weiyun; Zhang, Yongqing; Wang, Aiping; Liu, Yanhong
Molecular detection of bacterial pathogens based on LAMP methods is a faster and simpler approach than conventional culture methods. Although different LAMP-based methods for pathogenic bacterial detection are available, a systematic comparison of these different LAMP assays has not been performed. In this paper, we compared 12 in-house real-time LAMP assays with a commercialized kit (Isothermal Master Mix) for the detection of Listeria monocytogenes, Salmonella spp, Staphylococcus aureus, Escherichia coli O157, E. coli O26, E. coli O45, E. coli O103, E. coli O111, E. coli O121, E. coli O145 and Streptococcus agalactiae. False-positive results were observed in all 12 in-house real-time LAMP assays, while all the negative controls of Isothermal Master Mix remained negative after amplification. The detection limit of Isothermal Master Mix for Listeria monocytogenes, Salmonella spp, Staphylococcus aureus, Escherichia coli O157, E. coli O26, E. coli O45, E. coli O103, E. coli O111, E. coli O121 and Streptococcus agalactiae was 1 pg, whereas the sensitivity of the commercialized kit for E. coli O145 was 100 pg. In conclusion, the 12 in-house real-time LAMP assays were impractical to use, while the commercialized kit Isothermal Master Mix was useful for the detection of most bacterial pathogens.
Roberts, Christine C; Swoyer, Ryan; Bryan, Janine T; Taddeo, Frank J
Real-time human papillomavirus (HPV) type-specific multiplex PCR assays were developed to detect HPV DNA in specimens collected for the efficacy determination of the quadrivalent HPV (type 6, 11, 16, and 18) L1 virus-like particle (VLP) vaccine (Gardasil). We evaluated the concordance between type-specific multiplex HPV PCR and the widely used, commercially available Roche Linear Array genotyping PCR assay. Female genital swab specimens were tested for the presence of L1, E6, and E7 sequences of HPV type 6 (HPV6), HPV11, HPV16, HPV18, HPV31, HPV45, HPV52, and HPV58 and E6 and E7 sequences of HPV33, HPV35, HPV39, HPV51, HPV56, and HPV59 in type- and gene-specific real-time multiplex PCR assays. Specimens were also tested for the presence of L1 sequences using two versions of the Roche Linear Array genotyping assay. Measures of concordance of a modified version of the Linear Array and the standard Linear Array PCR assay were evaluated. With specimen DNA extraction using the Qiagen Spin blood kit held as the constant, multiplex PCR assays detect more HPV-positive specimens for the 14 HPV types common to both than either version of the Linear Array HPV genotyping assay. Type-specific agreements between the assays were good, at least 0.838, but were often driven by negative agreement in HPV types with low prevalence, as evidenced by reduced proportions of positive agreement. Overall HPV status agreements ranged from 0.615 for multiplex PCR and standard Linear Array to 0.881 for multiplex PCR and modified Linear Array. An alternate DNA extraction technique, that used by the Qiagen MinElute kit, impacted subsequent HPV detection in both the multiplex PCR and Linear Array assays.
Torres, Andrea N; O'Halloran, Kevin P; Larson, Laurie J; Schultz, Ronald D; Hoover, Edward A
We previously defined four categories of feline leukemia virus (FeLV) infection, designated as abortive, regressive, latent, and progressive. To determine if detectable viral DNA is transcriptionally active in the absence of antigenemia, we developed and validated a real-time viral RNA qPCR assay. This assay proved to be highly sensitive, specific, reproducible, and allowed reliable quantitation. We then applied this methodology, together with real-time DNA qPCR and p27 capsid antigen capture ELISA, to examine cats challenged with FeLV. We found that circulating viral RNA and DNA levels were highly correlated and the assays were almost in perfect agreement. This indicates that the vast majority of viral DNA is transcriptionally active, even in the absence of antigenemia. The real-time qPCR assays are more sensitive than the most commonly used FeLV diagnostic assay, the p27 capsid antigen capture ELISA. Application of qPCR assays may add greater depth in understanding of FeLV-host relationships.
Benitez, Alvaro J; Winchell, Jonas M
We developed a single tube multiplex real-time PCR assay that allows for the rapid detection and typing of 9 nonpneumophila Legionella spp. isolates that are clinically relevant. The multiplex assay is capable of simultaneously detecting and discriminating L. micdadei, L. bozemanii, L. dumoffii, L. longbeachae, L. feeleii, L. anisa, L. parisiensis, L. tucsonensis serogroup (sg) 1 and 3, and L. sainthelensis sg 1 and 2 isolates. Evaluation of the assay with nucleic acid from each of these species derived from both clinical and environmental isolates and typing strains demonstrated 100% sensitivity and 100% specificity when tested against 43 other Legionella spp. Typing of L. anisa, L. parisiensis, and L. tucsonensis sg 1 and 3 isolates was accomplished by developing a real-time PCR assay followed by high-resolution melt (HRM) analysis targeting the ssrA gene. Further typing of L. bozemanii, L. longbeachae, and L. feeleii isolates to the serogroup level was accomplished by developing a real-time PCR assay followed by HRM analysis targeting the mip gene. When used in conjunction with other currently available diagnostic tests, these assays may aid in rapidly identifying specific etiologies associated with Legionella outbreaks, clusters, sporadic cases, and potential environmental sources.
De Los Santos, Maxy; Soberón, Valeria; Lucas, Carmen M.; Matlashewski, Greg; Llanos-Cuentas, Alejandro; Ore, Marianela; Baldeviano, G. Christian; Edgel, Kimberly A.; Lescano, Andres G.; Graf, Paul C. F.; Bacon, David J.
In South America, various species of Leishmania are endemic and cause New World tegumentary leishmaniasis (NWTL). The correct identification of these species is critical for adequate clinical management and surveillance activities. We developed a real-time polymerase chain reaction (PCR) assay and evaluated its diagnostic performance using 64 archived parasite isolates and 192 prospectively identified samples collected from individuals with suspected leishmaniasis enrolled at two reference clinics in Lima, Peru. The real-time PCR assay was able to detect a single parasite and provided unambiguous melting peaks for five Leishmania species of the Viannia subgenus that are highly prevalent in South America: L. (V.) braziliensis, L. (V.) panamensis, L. (V.) guyanensis, L. (V.) peruviana and L. (V.) lainsoni. Using kinetoplastid DNA-based PCR as a gold standard, the real-time PCR had sensitivity and specificity values of 92% and 77%, respectively, which were significantly higher than those of conventional tests such as microscopy, culture and the leishmanin skin test (LST). In addition, the real-time PCR identified 147 different clinical samples at the species level, providing an overall agreement of 100% when compared to multilocus sequence typing (MLST) data performed on a subset of these samples. Furthermore, the real-time PCR was three times faster and five times less expensive when compared to PCR - MLST for species identification from clinical specimens. In summary, this new assay represents a cost-effective and reliable alternative for the identification of the main species causing NWTL in South America. PMID:23301111
Rossmanith, Peter; Wagner, Martin
The validation of quantitative real-time PCR systems and above all, proof of the detection limit of this method, is a frequently and intensively discussed topic in food pathogen detection. Among proper sample collection, assay design, careful experimental design, execution of real-time PCR, and data analysis, the validation of the method per se ensuring reliable quantification data is of prime importance. The purpose of this study was to evaluate a novel validation tool for real-time PCR assays, based on the theoretical possibility of the amplification of a single DNA target. The underlying mathematical basis for the work is Poisson distribution, which describes patterns of low particle numbers in a volume. In this context, we focused on the quantitative aspect of real-time PCR for the first time. This allowed for demonstration of the reliable amplification of a lone target DNA molecule and the demonstration of the distinct discrimination between integer molecular numbers when using low initial copy numbers. A real-time PCR assay amplifying a 274-bp fragment of the positive regulatory protein A locus of Listeria monocytogenes was used for this work. Evidence for a linear range of quantification from a single target copy to 10 ng of target DNA was experimentally demonstrated, and evidence for the significance of this novel validation approach is presented here.
Atienza, Josephine M; Yu, Naichen; Kirstein, Shelli L; Xi, Biao; Wang, Xiaobo; Xu, Xiao; Abassi, Yama A
Cell-based assays have become an integral part of the preclinical drug development process. Recently, noninvasive label-free cell-based assay technologies have taken center stage, offering important and distinct advantages over and in addition to traditional label-based endpoint assays. Dynamic monitoring of live cells, the preclusion of label, and kinetics are some of the fundamental features of cell-based label-free technologies. In this article we will discuss the real-time cell electronic sensing (RT-CES, ACEA Biosciences Inc., San Diego, CA) system and some of its key applications for cell-based assays such as cell proliferation and cytotoxicity, functional assays for receptor-ligand analysis, cell adhesion and spreading assays, dynamic monitoring of endothelial barrier function, and dynamic monitoring of cell migration and invasion. Also, where appropriate we will briefly discuss other label-free technologies in an application-specific manner.
A duplex quantitative real-time polymerase chain reaction (qPCR) assay was developed to differentiate between Bolbophorus damnificus and Bolbophorus type II species cercariae. Both trematode species are prevalent throughout the commercial catfish industry,.as both infect the ram’s horn snail, Plano...
Eischeid, Anne C; Kim, Bang-hyun; Kasko, Sasha M
Food allergen detection methods must be able to specifically detect minute quantities of an allergenic food in a complex food matrix. One technique that can be used is real-time PCR. For the work described here, real-time PCR assays were developed to detect penaeid shrimp and blue crab, crustacean shellfish allergens. The method was tested using shrimp meat and crab meat spiked into several types of foods, including canned soups, deli foods, meat, seafood, and prepared seafood products. Foods were spiked with either shrimp or crab at levels ranging from 0.1 to 10⁶ parts per million (ppm) and analyzed either raw or cooked by a variety of methods. Real-time PCR data were used to generate linear standard curves, and assays were evaluated with respect to linear range and reaction efficiency. Results indicate that both assays performed well in a variety of food types. High reaction efficiencies were achieved across a linear range of 6-8 orders of magnitude. Limits of detection were generally between 0.1 and 1 ppm. Cooking methods used to simulate thermal processing of foods had little effect on assay performance. This work demonstrates that real-time PCR can be a valuable tool in the detection of crustacean shellfish.
Real-time quantitative PCR assays that target the human-associated HF183 bacterial cluster are considered to be some of the top performing methods for the characterization of human fecal pollution in ambient surface waters. In response, the United States Environmental Protectio...
A subtype specific H7 real-time reverse transcriptase polymerase chain reaction (RT-PCR) assay developed by the Southeast Poultry Research Laboratory (SEPRL) for the detection of H7 in North and South American wild aquatic birds and poultry was validated as a collaborative effort by the SEPRL and Na...
Real-time quantitative PCR assays that target the human-associated HF183 bacterial cluster are considered to be some of the top performing methods for the characterization of human fecal pollution in ambient surface waters. In response, the United States Environmental Protectio...
Real-time quantitative PCR assays that target the human-associated HF183 bacterial cluster have been found to be some of the top performing methods for the characterization of human fecal pollution in ambient surface waters. The United States Environmental Protection Agency is planning to conduct a ...
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 ...
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...
Rodríguez, Alicia; Rodríguez, Mar; Luque, M Isabel; Martín, Alberto; Córdoba, Juan J
Aflatoxins are among the most toxic mycotoxins. Early detection and quantification of aflatoxin-producing species is crucial to improve food safety. In the present work, two protocols of real-time PCR (qPCR) based on SYBR Green and TaqMan were developed, and their sensitivity and specificity were evaluated. Primers and probes were designed from the o-methyltransferase gene (omt-1) involved in aflatoxin biosynthesis. Fifty-three mold strains representing aflatoxin producers and non-producers of different species, usually reported in food products, were used as references. All strains were tested for aflatoxins production by high-performance liquid chromatography-mass spectrometry (HPLC-MS). The functionality of the proposed qPCR method was demonstrated by the strong linear relationship of the standard curves constructed with the omt-1 gene copy number and Ct values for the different aflatoxin producers tested. The ability of the qPCR protocols to quantify aflatoxin-producing molds was evaluated in different artificially inoculated foods. A good linear correlation was obtained over the range 4 to 1 log cfu/g per reaction for all qPCR assays in the different food matrices (peanuts, spices and dry-fermented sausages). The detection limit in all inoculated foods ranged from 1 to 2 log cfu/g for SYBR Green and TaqMan assays. No significant effect was observed due to the different equipment, operator, and qPCR methodology used in the tests of repeatability and reproducibility for different foods. The proposed methods quantified with high efficiency the fungal load in foods. These qPCR protocols are proposed for use to quantify aflatoxin-producing molds in food products. Copyright © 2012 Elsevier Ltd. All rights reserved.
Favaro, Marco; Savini, Vincenzo; Favalli, Cartesio; Fontana, Carla
A central nervous system (CNS) infection, such as meningitis, is a serious and life-threatening condition. Bacterial meningitis can be severe and may result in brain damage, disability or even death. Rapid diagnosis of CNS infections and identification of the pathogenic microorganisms are needed to improve the patient outcome. Bacterial culture of a patient's cerebrospinal fluid (CSF) is currently considered the "gold standard" for diagnosing bacterial meningitis. From the CSF cultures researchers can assess the in vitro susceptibility of the causative microorganism to determine the best antibiotic treatment. However, many of the culture assays, such as microscopy and the latex agglutination test are not sensitive. To enhance pathogen detection in CSF samples we developed a multi-target real-time PCR assay that can rapidly identify six different microorganisms: Streptococcus pneumoniae, Neisseria meningitidis, Haemophilus influenzae, Streptococcus agalactiae, Listeria monocytogenes and Cryptococcus neoformans. In this study we applied this PCR analysis to 296 CSF samples from patients who were suspected of having meningitis. Of the 296 samples that were examined, 59 samples were positive according to the CSF culture and/or molecular assays. Forty-six CSF samples were positive for both the CSF culture and our real-time PCR assay, while 13 samples were positive for the real-time PCR but negative for the traditional assays. This discrepancy may have been caused by the fact that these samples were collected from 23 patients who were treated with antimicrobials before CSF sampling.
Naderi, Mahmood; Abdul Tehrani, Hossein; Soleimani, Masoud; Shabani, Iman; Hashemi, Seyed Mahmoud
miR-122 is a liver-specific miRNA that has significant gene expression alterations in response to specific pathophysiological circumstances of liver such as drug-induced liver injury, hepatocellular carcinoma, and hepatitis B and C virus infections. Therefore, accurate and precise quantification of miR-122 is very important for clinical diagnostics. However, because of the lack of in vitro diagnostics assays for miR-122 detection and quantification of the existence of an open-source assay could inevitably provide external evaluation by other researchers and the chance of promoting the assay when required. The aim of this study was to develop a Taqman real-time polymerase chain reaction assay, which is capable of robust and reliable quantification of miR-122 in different sample types. We used stem loop methodology to design a specific Taqman real-time polymerase chain reaction assay for miR-122. This technique enabled us to reliably and reproducibly quantify short-length oligonucleotides such as miR-122. The specificity, sensitivity, interassay and intra-assay, and the dynamic range of the assay were experimentally determined by their respective methodology. The assay had a linear dynamic range of 3E to 4.8E miR-122 copies/reaction and the limit of detection was determined to be between 960 and 192 copies/reaction with 95% confidence interval. The assay gave a coefficient of variation for the Ct values of <1.4% and 0.78% for intra-assay and interassay, respectively. Taking into account that miR-122 is expressed in >50,000 copies per hepatocyte, this assay is able to suffice the need for reliable detection and quantification of this miRNA. Therefore, this study can be considered as a start point for standardizing miR-122 quantification.
Mehta, Maitry S; McClure, J T; Mangold, Kathy; Peterson, Lance R
We compared 3 real-time PCR assays: off-label use of 2 commercial assays (BD-GeneOhm™ MRSA assay for methicillin-resistant Staphylococcus aureus [MRSA] detection and BD-GeneOhm StaphSR™ for MRSA and methicillin-susceptible S. aureus detection) and an in-house real-time PCR assay for detection of total S. aureus from clinical specimens. Testing was performed on 200 distinct specimens. Sensitivity, specificity, positive predictive value, and negative predictive value were calculated using culture as the gold standard. The prevalence of S. aureus in the samples was 44.5%, and MRSA was 20%. For total S. aureus, the StaphSR-PCR and the in-house PCR assays had a sensitivity and specificity of 94.4% and 96.4% and 93.3% and 99.1%, respectively. For MRSA detection, the StaphSR and the BD GeneOhm assay had a sensitivity and specificity of 92.5% and 98.8% and 92.5% and 96.3%, respectively. This study demonstrates the potential use of tests like the StaphSR-PCR assay for rapid detection of S. aureus and MRSA directly from clinical specimens; however, culture follow-up would be needed to identify other potential pathogens in the specimen.
Hagen, Ralf Matthias; Hinz, Rebecca; Tannich, Egbert; Frickmann, Hagen
We compared the performance of an in-house and a commercial malaria polymerase chain reaction (PCR) assay using freeze-thawed hemolytic blood samples. A total of 116 freeze-thawed ethylenediamine tetraacetic acid (EDTA) blood samples of patients with suspicion of malaria were analyzed by an in-house as well as by a commercially available real-time PCR. Concordant malaria negative PCR results were reported for 39 samples and malaria-positive PCR results for 67 samples. The in-house assay further detected one case of Plasmodium falciparum infection, which was negative in the commercial assay as well as five cases of P. falciparum malaria and three cases of Plasmodium vivax malaria, which showed sample inhibition in the commercial assay. The commercial malaria assay was positive in spite of a negative in-house PCR result in one case. In all concordant results, cycle threshold values of P. falciparum-positive samples were lower in the commercial PCR than in the in-house assay. Although Ct values of the commercial PCR kit suggest higher sensitivity in case of concordant results, it is prone to inhibition if it is applied to hemolytic freeze-thawed blood samples. The number of misidentifications was, however, identical for both real-time PCR assays.
Rashid, Ridwan Bin; Ferdous, Jannatul; Tulsiani, Suhella; Jensen, Peter Kjaer Mackie; Begum, Anowara
Vibrio cholerae O1 and O139 has been known for its ability to cause epidemics. These strains produce cholera toxin which is the main cause of secretory diarrhea. V. cholerae non-O1 and non-O139 strains are also capable of causing gastroenteritis as well as septicemia and peritonitis. It has been proven that virulence factors such as T6SS, hapA, rtxA, and hlyA are present in almost all V. cholerae strains. It is imperative that viable but non-culturable cells of V. cholerae are also detected since they are also known to cause diarrhea. Thus, the aim of this study was to develop an assay that detects all V. cholerae regardless of their serotype, culturable state, and virulence genes present, by targeting the species specific conserved ompW sequence. The developed assay meets these goals with 100% specificity and is capable of detecting as low as 5.46 copy number of V. cholerae. Detection is rapid since neither lengthy incubation period nor electrophoresis is required. The assay had excellent repeatability (CV%: 0.24-1.32) and remarkable reproducibility (CV%: 1.08-3.7). Amplification efficiencies in the 89-100% range were observed. The assay is more economical than Taqman-based multiplex real-time PCR assays. Compared to other real-time assays, the ompW assay is specific and sensitive, has better repeatability and reproducibility, and is more economical.
Kim, Nack Joo; Youn, Minsang; Kim, Yongwook; Sung, Yeolmin; Kim, Dohoon
A method of detecting lead was developed using square wave anodic stripping voltammetry (SWASV) with DNA-carbon nanotube paste electrode (CNTPE). The results indicated a sensitive oxidation peak current of lead on the DNA-CNTPE. The curves were obtained within a concentration range of 50 ngL−1-20 mgL−1 with preconcentration time of 100, 200, and 400 sec at the concentration of mgL−1, μgL−1, and ngL−1, respectively. The observed relative standard deviation was 0.101% (n = 12) in the lead concentration of 30.0 μgL−1 under optimum conditions. The low detection limit (S/N) was pegged at 8 ngL−1 (2.6 × 10−8 M). Results showed that the developed method can be used in real-time assay in vivo without requiring any pretreatment and pharmaceutical samples, and food samples, as well as other materials requiring water source contamination analyses. PMID:24578800
Nabavi Zadeh, Pegah S; Mallak, Kassam Abdel; Carlsson, Nils; Åkerman, Björn
Mesoporous silica particles are used as support material for immobilization of enzymes. Here we investigated a fluorescence-based assay for real-time monitoring of the immobilization of lipase, bovine serum albumin, and glucose oxidase into micrometer-sized mesoporous silica particles. The proteins are labeled with the dye epicocconone, and the interaction with the particles is observed as an increase in emission intensity of the protein-dye conjugates that can be quantified if correcting for a comparatively slow photobleaching. The immobilization occurs in tens of minutes to hours depending on particle concentration and type of protein. In the limit of excess particles over proteins, the formation of the particle-protein complexes can be described by a single exponential growth for all three investigated proteins, and the fitted pseudo-first-order rate constant increases linearly with particle concentration for each protein type. The derived second-order rate constant k varies with the protein hydrodynamic radius according to k∼RH(-4.70±0.01), indicating that the rate-limiting step at high particle concentrations is not the diffusional encounter between proteins and particles but rather the entry into the pores, consistent with the hydrodynamic radii of the three proteins being smaller but comparable to the pore radius of the particles.
Waggoner, Jesse J.; Abeynayake, Janaki; Sahoo, Malaya K.; Gresh, Lionel; Tellez, Yolanda; Gonzalez, Karla; Ballesteros, Gabriela; Balmaseda, Angel; Karunaratne, Kumudu; Harris, Eva
A number of diagnostic tests are available for dengue virus (DENV) detection, including a variety of nucleic acid amplification tests (NAATs). However, reports describing a direct comparison of different NAATs have been limited. In this study, we report the design of an internally controlled real-time reverse transcriptase PCR (rRT-PCR) that detects all four DENV serotypes but does not distinguish between them (the pan-DENV assay). Two hundred clinical samples were then tested using four different DENV RT-PCR assays: the pan-DENV assay, a commercially produced, internally controlled DENV rRT-PCR (the Altona assay), a widely used heminested RT-PCR, and a serotype-specific multiplex rRT-PCR assay. The pan-DENV assay had a linear range extending from 1.0 to 7.0 log10 cDNA equivalents/μl and a lower limit of 95% detection ranging from 1.7 to 7.6 cDNA equivalents/μl, depending on the serotype. When measured against a composite reference standard, the pan-DENV assay proved to be more clinically sensitive than either the Altona or heminested assays, with a sensitivity of 98.0% compared to 72.3% and 78.8%, respectively (P ≤ 0.0001 for both comparisons). The pan-DENV assay detected DENV in significantly more samples collected on or after day 5 of illness and in a subgroup of patients with detectable anti-DENV IgM at presentation. No significant difference in sensitivity was observed between the pan-DENV assay and the multiplex rRT-PCR, despite the presence of an internal control in the former. The detection of DENV RNA late in the course of clinical illness should serve to lengthen the period during which a confirmed molecular diagnosis of DENV infection can be provided. PMID:23637298
Baris, Ibrahim; Etlik, Ozdal; Koksal, Vedat; Ocak, Zeynep; Baris, Saniye Tugba
Single-nucleotide polymorphism (SNP) genotyping is widely used in genetic association studies to characterize genetic factors underlying inherited traits. Despite many recent advances in high-throughput SNP genotyping, inexpensive and flexible methods with reasonable throughput levels are still needed. Real-time PCR methods for discovering and genotyping SNPs are becoming increasingly important in various fields of biology. In this study, we introduce a new, single-tube strategy that combines the tetra-primer ARMS PCR assay, SYBR Green I-based real-time PCR, and melting-point analysis with primer design strategies to detect the SNP of interest. This assay, T-Plex real-time PCR, is based on the T(m) discrimination of the amplified allele-specific amplicons in a single tube. The specificity, sensitivity, and robustness of the assay were evaluated for common mutations in the FV, PII, MTHFR, and FGFR3 genes. We believe that T-Plex real-time PCR would be a useful alternative for either individual genotyping requests or large epidemiological studies.
Jalal, Hamid; Al-Suwaine, Abdulrahman; Stephen, Hannah; Carne, Christopher; Sonnex, Christopher
This study investigated the comparative performance of the Amplicor assay and an in-house semi-automated, multiplex real-time PCR for the diagnosis of genital chlamydial infection. Four different assays, the COBAS Amplicor CT test (Amplicor PCR), in-house real-time PCR (IHRT-PCR), in-house nested cryptic plasmid PCR and in-house nested major outer membrane protein PCR, were performed on genital swabs from 1000 consecutive patients attending a genitourinary medicine clinic. The samples were designated true positive if Chlamydia trachomatis DNA was detected by at least two of the four above-mentioned assays while a sample was defined as true negative if C. trachomatis DNA was detected in only one or none of the assays. By this criterion, there were 129 true positive and 871 true negative samples for C. trachomatis DNA in this cohort. Amplicor PCR designated 144 samples positive: 128 (89%) of 144 samples were true positive and 16 (11%) were false positive. IHRT-PCR detected 126 of 129 true positive samples and did not generate any false positive results. The sensitivity of IHRT-PCR was comparable with, and specificity was higher than, Amplicor PCR for the diagnosis of genital chlamydial infection.
perfringens 13124 1000 0/2 0/2 Francisella tularensis NA 1000 0/2 0/2 Haemophlius influenzae 10211 1000 0/2 0/2 Listeia monocytogenes 15313 1000 0/2 0/2...Loveless BM, Norwood D, Zwiers SH, Mucker E, Hartmann C, et al. Smallpox and pan-orthopox virus detection by real-time 3 0-minor groove binder TaqMan ...Whitehouse CA, Hartmann C, Mucker E, et al. Monkeypox virus detection in rodents using real-time 3 0-minor groove binder TaqMan assays on the Roche
Watzinger, F; Suda, M; Preuner, S; Baumgartinger, R; Ebner, K; Baskova, L; Niesters, H G M; Lawitschka, A; Lion, T
A panel of 23 real-time PCR assays based on TaqMan technology has been developed for the detection and monitoring of 16 different viruses and virus families including human polyomaviruses BK virus and JC virus, human herpesviruses 6, 7, and 8, human adenoviruses, herpes simplex viruses 1 and 2, varicella-zoster virus, cytomegalovirus, Epstein-Barr virus, parvovirus B19, influenza A and B viruses, parainfluenza viruses 1 to 3, enteroviruses, and respiratory syncytial virus. The test systems presented have a broad dynamic range and display high sensitivity, reproducibility, and specificity. Moreover, the assays allow precise quantification of viral load in a variety of clinical specimens. The ability to use uniform PCR conditions for all assays permits simultaneous processing and detection of many different viruses, thus economizing the diagnostic work. Our observations based on more than 50,000 assays reveal the potential of the real-time PCR tests to facilitate early diagnosis of infection and to monitor the kinetics of viral proliferation and the response to treatment. We demonstrate that, in immunosuppressed patients with invasive virus infections, surveillance by the assays described may permit detection of increasing viral load several days to weeks prior to the onset of clinical symptoms. In virus infections for which specific treatment is available, the quantitative PCR assays presented provide reliable diagnostic tools for timely initiation of appropriate therapy and for rapid assessment of the efficacy of antiviral treatment strategies.
Zeller, Iris; Schabereiter-Gurtner, Claudia; Mihalits, Verena; Selitsch, Brigitte; Barousch, Wolfgang; Hirschl, Alexander M; Makristathis, Athanasios; Willinger, Birgit
The rise in the incidence of fungal infections and the expanding spectrum of fungal pathogens make early and broad detection of fungal pathogens essential. In the present study, a panfungal real-time PCR assay for the broad-range detection of fungal DNA (Fungi assay) in a wide variety of clinical specimens was developed. Our in-house, HybProbe real-time PCR assay targets the ITS2 region of fungal DNA. The applicability was evaluated by testing 105 clinical samples from 98 patients with suspected fungal infection. Samples included tissue biopsies, paraffin embedded tissues, aspirates, EDTA-anticoagulated blood, cerebrospinal fluids and bronchoalveolar lavages. Fungal pathogens were identified by the Fungi assay in 47 samples. In all of these cases, conventional methods and clinical data were also indicative for a fungal infection. Five samples were interpreted false negative. blast analyses of the amplicons derived from 11 samples revealed the presence of environmental fungal species while other tests and clinical data did not suggest a fungal infection. This fact might indicate contaminated samples. The remaining 42 samples were negative by the Fungi assay as well as the conventional methods and were therefore regarded as true negatives. Thus, sensitivity was 90.4 % and specificity 79.2 %. The Fungi assay improved the targeted diagnosis of fungal infections allowing pathogen identification in samples that were histologically positive but culture negative. For reliable diagnosis, results have to be interpreted in context with conventional methods and clinical data.
Li, Na; Neumann, Norman F; Ruecker, Norma; Alderisio, Kerri A; Sturbaum, Gregory D; Villegas, Eric N; Chalmers, Rachel; Monis, Paul; Feng, Yaoyu; Xiao, Lihua
The occurrence of Cryptosporidium oocysts in drinking source water can present a serious public health risk. To rapidly and effectively assess the source and human-infective potential of Cryptosporidium oocysts in water, sensitive detection and correct identification of oocysts to the species level (genotyping) are essential. In this study, we developed three real-time PCR genotyping assays, two targeting the small-subunit (SSU) rRNA gene (18S-LC1 and 18S-LC2 assays) and one targeting the 90-kDa heat shock protein (hsp90) gene (hsp90 assay), and evaluated the sensitivity and Cryptosporidium species detection range of these assays. Using fluorescence resonance energy transfer probes and melt curve analysis, the 18S-LC1 and hsp90 assays could differentiate common human-pathogenic species (C. parvum, C. hominis, and C. meleagridis), while the 18S-LC2 assay was able to differentiate nonpathogenic species (such as C. andersoni) from human-pathogenic ones commonly found in source water. In sensitivity evaluations, the 18S-LC2 and hsp90 genotyping assays could detect as few as 1 Cryptosporidium oocyst per sample. Thus, the 18S-LC2 and hsp90 genotyping assays might be used in environmental monitoring, whereas the 18S-LC1 genotyping assay could be useful for genotyping Cryptosporidium spp. in clinical specimens or wastewater samples.
Kokkula, Chakradhar; Palanisamy, Navaneethan; Ericstam, Malin; Lennerstrand, Johan
There are arrays of in vitro assays to quantify the activity of HIV-1 reverse transcriptase (HIV-1 RT). These assays utilize either chemically customized/labelled nucleotides, or TaqMan probes, or radiolabeled nucleotides/primers. Although several real-time PCR assays exist commercially for measuring the RT activity, which are usually used for quantifying the viral titres, these assays are not optimized for measuring the inhibitory concentrations (IC50) of HIV-1 RT inhibitors. Moreover, a recently established inorganic pyrophosphate-coupled enzyme assay cannot be employed for studying nonphosphorylated nucleoside reverse transcriptase inhibitors (NRTIs). In the present study, we have developed a novel one-step assay with native nucleotide substrates and SYBR Green II dye to determine IC50 values of triphosphorylated NRTIs against HIV-1 RT. Using exact batches of wild-type and mutant RT, and triphosphorylated NRTIs, we showed that our method gave IC50 values for inhibitors similar to that of an earlier published colorimetric assay with BrdUTP substrate (CABS). Our assay should be suitable for high-throughput screening of antiretroviral drugs and could also be suitable for studying drug resistance profiles. Additionally, we also used our assay to study inhibition by AZT in its nonphosphorylated form by supplementing the reaction mixture with necessary kinases and ATP.
Li, Na; Neumann, Norman F.; Ruecker, Norma; Alderisio, Kerri A.; Sturbaum, Gregory D.; Villegas, Eric N.; Chalmers, Rachel; Monis, Paul; Feng, Yaoyu
The occurrence of Cryptosporidium oocysts in drinking source water can present a serious public health risk. To rapidly and effectively assess the source and human-infective potential of Cryptosporidium oocysts in water, sensitive detection and correct identification of oocysts to the species level (genotyping) are essential. In this study, we developed three real-time PCR genotyping assays, two targeting the small-subunit (SSU) rRNA gene (18S-LC1 and 18S-LC2 assays) and one targeting the 90-kDa heat shock protein (hsp90) gene (hsp90 assay), and evaluated the sensitivity and Cryptosporidium species detection range of these assays. Using fluorescence resonance energy transfer probes and melt curve analysis, the 18S-LC1 and hsp90 assays could differentiate common human-pathogenic species (C. parvum, C. hominis, and C. meleagridis), while the 18S-LC2 assay was able to differentiate nonpathogenic species (such as C. andersoni) from human-pathogenic ones commonly found in source water. In sensitivity evaluations, the 18S-LC2 and hsp90 genotyping assays could detect as few as 1 Cryptosporidium oocyst per sample. Thus, the 18S-LC2 and hsp90 genotyping assays might be used in environmental monitoring, whereas the 18S-LC1 genotyping assay could be useful for genotyping Cryptosporidium spp. in clinical specimens or wastewater samples. PMID:26092455
Melendez, J H; Frankel, Y M; An, A T; Williams, L; Price, L B; Wang, N-Y; Lazarus, G S; Zenilman, J M
Chronic wounds cause substantial morbidity and disability. Infection in chronic wounds is clinically defined by routine culture methods that can take several days to obtain a final result, and may not fully describe the community of organisms or biome within these wounds. Molecular diagnostic approaches offer promise for a more rapid and complete assessment. We report the development of a suite of real-time PCR assays for rapid identification of bacteria directly from tissue samples. The panel of assays targets 14 common, clinically relevant, aerobic pathogens and demonstrates a high degree of sensitivity and specificity using a panel of organisms commonly associated with chronic wound infection. Thirty-nine tissue samples from 29 chronic wounds were evaluated and the results compared with those obtained by culture. As revealed by culture and PCR, the most common organisms were methicillin-resistant Staphylococcus aureus (MRSA) followed by Streptococcus agalactiae (Group B streptococcus) and Pseudomonas aeruginosa. The sensitivities of the PCR assays were 100% and 90% when quantitative and qualitative culture results were used as the reference standard, respectively. The assays allowed the identification of bacterial DNA from ten additional organisms that were not revealed by quantitative or qualitative cultures. Under optimal conditions, the turnaround time for PCR results is as short as 4-6 h. Real-time PCR is a rapid and inexpensive approach that can be easily introduced into clinical practice for detection of organisms directly from tissue samples. Characterization of the anaerobic microflora by real-time PCR of chronic wounds is warranted.
Taylor, Ninon; Grabmeier-Pfistershammer, Katharina; Egle, Alexander; Greil, Richard; Rieger, Armin; Ledergerber, Bruno; Oberkofler, Hannes
High-sensitive real-time PCR assays are routinely used to monitor HIV-1 infected subjects. Inter-assay discrepancies have been described at the low viral load (VL) end, where clinical decisions regarding possible virological rebound are based. A retrospective study was performed to analyze frequencies of viral blips after transition to the COBAS Ampliprep/COBAS TaqMan v2.0 HIV-1 assay (Taqman v2.0) in patients with prior undetectable VLs as measured with the Roche Cobas Ampliprep Amplicor HIV-1 Monitor Test, v1.5 (Amplicor) and was evaluated in comparison to a group of patients monitored with the Abbott Real-time HIV-1 assay (Abbott RT) during the same period of time. 85 of 373 patients with VLs below the limit of quantification with Amplicor had VLs >50 copies/mL after transition to the TaqMan v2.0 assay. Among these 74.1% had VLs ranging from 50-499 copies/mL, 22.9% had VLs >500 copies/mL. From 22 patients with initial Taqman v2.0 based VLs exceeding 500 copies/mL, 6 patients had VLs <20 copies/mL after novel VL measurement on a next visit. In our control group with VL quantification using the Abbott RT assay, only 1 patient became detectable and showed a VL of <40 copies/mL after new measurement. Transition to the Taqman v2.0 assay was accompanied by an increase of quantifiable HIV-1 VLs in patients with long term viral suppression under antiretroviral therapy that might be attributed to technical shortcomings of the Taqman v2.0 assay. A high test variability at the low VL end but also beyond was observed, making meaningful clinical interpretation of viral blips derived from different assays difficult.
Anderson, Trevor P; Werno, Anja M; Barratt, Kevin; Mahagamasekera, Patalee; Murdoch, David R; Jennings, Lance C
Multiplex PCR has become the test of choice for the detection of multiple respiratory viruses in clinical specimens. However, there are few direct comparisons of different PCR assays. This study compares 4 different multiplex PCR assays for the recovery of common respiratory viruses. We tested 213 respiratory specimens using four different multiplex PCR assays: the xTAG respiratory viral panel fast (Abbott Molecular Laboratories), Fast-track Respiratory Pathogen assay (Fast-track Diagnostics), Easyplex respiratory pathogen 12 kit (Ausdiagnostics), and an in-house multiplex real-time PCR assay. The performance of the four assays was very similar, with 93-100% agreement for all comparisons. Other issues, such as through-put, technical requirements and cost, are likely to be as important for making a decision about which of these assays to use given their comparative performance.
Rosadas, Carolina; Cabral-Castro, Mauro Jorge; Vicente, Ana Carolina Paulo; Peralta, José Mauro; Puccioni-Sohler, Marzia
The objective of this study was to validate a TaqMan real-time PCR assay for HTLV-1 proviral load detection in peripheral blood mononuclear cells. TARL-2 cells were used to generate a standard curve. Peripheral blood mononuclear cell gDNA from 27 seropositive and 23 seronegative samples was analyzed. The sensitivity, specificity, accuracy, precision, dynamic range of the standard curve and qPCR efficiency were evaluated. All of the positive samples amplified the target gene. All of the negative samples amplified only the control gene (β-actin). The assay presented 100% specificity and sensibility. The intra- and inter-assay variability was 2.4% and 2.2%, respectively. The qPCR efficiency, slope and correlation coefficients (r2) were all acceptable. The limit of detection was 1 copy/rxn. This assay can reliably quantify HTLV-1 proviral load. Copyright © 2013 Elsevier B.V. All rights reserved.
Dr Maude E Abbott (1869 to 1940) is the only Canadian and the only woman represented in Diego Rivera's great mural of the History of Cardiology in Mexico City. She gained this place among the world's famous physicians and scientists by her outstanding studies of congenital heart disease. Her atlas of 1000 cases with clinical, pathological and morphological findings is the first systematic study of these anomalies. Dr Abbott developed a pathophysiological classification of cardiovascular defects fundamental for the development of cardiac surgery. She also considered prevention by prenatal care, recognizing possible genetic and environmental risk factors. Maude Abbott was a thoughtful clinician and a brilliant scientist of incomparable industry. She leaves an unfinished legacy to make the prevention of congenital heart disease a reality.
Eigner, U; Hiergeist, A; Veldenzer, A; Rohlfs, M; Schwarz, R; Holfelder, M
Diarrheagenic E. coli (DEC) are one of the most common causes for diarrhea worldwide, especially in children. We evaluated the rapid RIDA ® GENE (RG) real-time multiplex PCR assays (R-Biopharm, Darmstadt, Germany) for the detection of the most important diarrheagenic E. coli. Three hundred fifteen liquid or non-formed stool specimens were examined. The results of the RG multiplex assays were compared to specific PCR methods. The sensitivity and specificity of the RG PCRs were as follows, 100%/100% for the detection of EHEC, 96.3% and 99% for EPEC, 100% and 100% for the detection of EAEC, ETEC and EIEC, respectively. Overall, the RG real-time PCR system for the detection of DEC tested in this study provided reliable and rapid results and shows the ability as a useful addendum for the detection of diarrheagenic E. coli in the medical laboratory.
Elkins, Kelly M; Perez, Anjelica C U; Sweetin, Katherine C
We demonstrate a method for developing real-time polymerase chain reaction (PCR) high-resolution melt (HRM) assays to identify multiple species present in a mixture simultaneously using LCGreen Plus and melt temperatures. Highly specific PCR primers are designed to yield amplicons with different melt temperatures for simple routine species identification compared with differentiating melt curve kinetics traces or difference plots. This method is robust and automatable, and it leads to savings in time and reagent costs, is easily modified to probe any species of interest, eliminates the need for post-PCR gel or capillary electrophoresis in routine assays, and requires no expensive dye-labeled primers.
Background The tick-borne apicomplexan bovine parasite Theileria annulata is endemic in many tropical and temperate areas, including Minorca (Balearic Islands, Spain). Real-time PCR is widely used for the detection of piroplasms but quantification is not commonly considered. Results We developed a real-time quantitative PCR (qPCR) assay for the detection and quantification of T. annulata that included an internal amplification control (IAC) to monitor for the presence of potential inhibitors. Specificity, sensitivity, precision, linear range and PCR efficiency were calculated and different methods for transformation of quantification cycle (Cq) values into quantities (Q) were evaluated. The assay was able to detect (100% probability) and quantify (linear response) 100 gene copies, and clinical sensitivity was set at 10 T. annulata per μl of blood. The assay was then validated on 141 bovine blood samples analyzed in parallel by a Luminex® suspension array, showing the utility of the qPCR assay developed here for the detection and quantification of the parasite in field conditions. Once validated it was used to monitor T. annulata parasitaemia throughout a year in 8 carrier animals from a farm in Minorca. Conclusions The developed qPCR assay offers a reliable and simple way to quantify T. annulata infection loads, which could prove crucial in studying the role of carrier animals as a source of the infection, or assessing the efficacy of treatment and control measures. PMID:22889141
characterization of highly pathogenic viruses : application during Crimean-Congo 313 haemorrhagic fever virus outbreaks in Eastern Europe and the Middle East...1 Sequence optimized real-time RT-PCR assay for detection of Crimean-Congo hemorrhagic fever 1 virus 2 3 JW Koehler1, KL Delp1, AT Hall1, SP...Institute of Infectious Diseases, 1425 Porter 9 Street, Fort Detrick, MD, 21702 USA 10 11 12 Abstract 13 14 Crimean-Congo hemorrhagic fever virus
Bergmans, Anneke M C; Rossen, John W A
Bartonella henselae is the causative agent of cat-scratch disease (CSD), usually presenting itself as a -self-limiting lymphadenopathy. In this chapter an internally controlled Taqman probe-based real-time PCR targeting the groEL gene of Bartonella spp. is described. This assay allows for the rapid, sensitive, and simple detection of Bartonella spp. in samples from CSD or endocarditis suspects, and it is suitable for implementation in the diagnostic microbiology laboratory.
Robène, Isabelle; Perret, Marion; Jouen, Emmanuel; Escalon, Aline; Maillot, Marie-Véronique; Chabirand, Aude; Moreau, Aurélie; Laurent, Annie; Chiroleu, Frédéric; Pruvost, Olivier
Bacterial blight of onion is an emerging disease threatening world onion production. The causal agent Xanthomonas axonopodis pv. allii is seed transmitted and a reliable and sensitive tool is needed to monitor seed exchanges. A triplex quantitative real-time PCR assay was developed targeting two X. axonopodis pv. allii-specific markers and an internal control chosen in 5.8S rRNA gene from Alliaceae. Amplification of at least one marker indicates the presence of the bacterium in seed extracts. This real-time PCR assay detected all the 79 X. axonopodis pv. allii strains tested and excluded 85.2% of the 135 non-target strains and particularly all 39 saprophytic and pathogenic bacteria associated with onion. Cross-reactions were mainly obtained for strains assigned to nine phylogenetically related X. axonopodis pathovars. The cycle cut-off was estimated statistically at 36.3 considering a risk of false positive of 1%. The limit of detection obtained in at least 95% of the time (LOD 95%) was 5×10(3) CFU/g (colony forming unit/g). The sensitivity threshold was found to be 1 infected seed in 32,790 seeds. This real-time PCR assay should be useful for preventing the long-distance spread of X. axonopodis pv. allii via contaminated seed lots and determining the epidemiology of the bacterium.
Pegels, Nicolette; González, Isabel; García, Teresa; Martín, Rosario
A highly sensitive TaqMan real-time PCR assay targeting the mitochondrial 12S rRNA gene was developed for detection of an avian-specific DNA fragment (68bp) in farm animal and pet feeds. The specificity of the assay was verified against a wide representation of animal and plant species. Applicability assessment of the avian real-time PCR was conducted through representative analysis of two types of compound feeds: industrial farm animal feeds (n=60) subjected to extreme temperatures, and commercial dog and cat feeds (n=210). Results obtained demonstrated the suitability of the real-time PCR assay to detect the presence of low percentages of highly processed avian material in the feed samples analysed. Although quantification results were well reproducible under the experimental conditions tested, an accurate estimation of the target content in feeds is impossible in practice. Nevertheless, the method may be useful as an alternative tool for traceability purposes within the framework of feed control. Copyright © 2013 Elsevier Ltd. All rights reserved.
Randhawa, Gurinder Jit; Chhabra, Rashmi; Bhoge, Rajesh K; Singh, Monika
Bt cotton events MON531 and MON15985 are authorized for commercial cultivation in more than 18 countries. In India, four Bt cotton events have been commercialized; more than 95% of total area under genetically modified (GM) cotton cultivation comprises events MON531 and MON15985. The present study reports on the development of efficient event-specific visual and real-time loop-mediated isothermal amplification (LAMP) assays for detection and identification of cotton events MON531 and MON15985. Efficiency of LAMP assays was compared with conventional and real-time PCR assays. Real-time LAMP assay was found time-efficient and most sensitive, detecting up to two target copies within 35 min. The developed real-time LAMP assays, when combined with efficient DNA extraction kit/protocol, may facilitate onsite GM detection to check authenticity of Bt cotton seeds.
Dunbar, N L; Bruggink, L D; Marshall, J A
The current study examined the efficacy of the RIDAGENE norovirus (NoV) real-time polymerase chain reaction assay (R-Biopharm, Darmstadt, Germany) for use in a routine diagnostic laboratory. The RIDAGENE assay had an overall sensitivity of 98% but was more sensitive for GII than GI NoV. The assay had a specificity of 98%. The RIDAGENE assay could detect a variety of GI and GII open reading frame 2 genotypes including GI.1, GI.3, GI.8, GI.13, GII.2, GII.3, GII.4 (including the following variants: 2006b, 2009, 2012, and 3 others that have not been assigned), GII.6, GII.12, and GII.13. The assay did not cross react with a number of gastroenteritis viruses including adenovirus, astrovirus, rotavirus, and sapovirus. The assay was straightforward to perform, and for a run of 50 specimens, a result was obtainable in roughly 4 hours. The RIDAGENE assay can be recommended as a valuable detection method for NoV. Copyright © 2014 Elsevier Inc. All rights reserved.
Bierbaum, Sibylle; Forster, Johannes; Berner, Reinhard; Rücker, Gerta; Rohde, Gernot; Neumann-Haefelin, Dieter; Panning, Marcus
The aim of this study was to determine the prevalence of respiratory viruses and to prospectively evaluate the performance of the fast-track diagnostics (FTD) respiratory pathogens multiplex PCR assay shortly after the 2009/10 influenza pandemic. Highly sensitive monoplex real-time PCR assays served as references. Discrepant results were further analyzed by the xTAG RVP Fast assay. A total of 369 respiratory samples from children and adults were collected prospectively in Germany from December 2009 until June 2010. The sensitivity and specificity of the FTD assay after resolution of discrepant results was 92.2 % and 99.5 %, respectively. Lowest specificity of the FTD assay was observed for human bocavirus. Multiple detections were recorded in 33/369 (8.9 %) of the samples by monoplex PCR and in 43/369 (11.7 %) using the FTD assay. The most prevalent viruses were respiratory syncytial virus and human metapneumovirus. Only pandemic influenza virus A/H1N1 (2009), and not seasonal influenza virus, was detected. Viruses other than influenza virus accounted for the majority of acute respiratory infections. The FTD assay can be easily implemented in general diagnostic laboratories and facilitate the optimization of patient-management schemes.
Green, Hyatt C; Haugland, Richard A; Varma, Manju; Millen, Hana T; Borchardt, Mark A; Field, Katharine G; Walters, William A; Knight, R; Sivaganesan, Mano; Kelty, Catherine A; Shanks, Orin C
Quantitative real-time PCR (qPCR) assays that target the human-associated HF183 bacterial cluster within members of the genus Bacteroides are among the most widely used methods for the characterization of human fecal pollution in ambient surface waters. In this study, we show that a current TaqMan HF183 qPCR assay (HF183/BFDrev) routinely forms nonspecific amplification products and introduce a modified TaqMan assay (HF183/BacR287) that alleviates this problem. The performance of each qPCR assay was compared in head-to-head experiments investigating limits of detection, analytical precision, predicted hybridization to 16S rRNA gene sequences from a reference database, and relative marker concentrations in fecal and sewage samples. The performance of the modified HF183/BacR287 assay is equal to or improves upon that of the original HF183/BFDrev assay. In addition, a qPCR chemistry designed to combat amplification inhibition and a multiplexed internal amplification control are included. In light of the expanding use of PCR-based methods that rely on the detection of extremely low concentrations of DNA template, such as qPCR and digital PCR, the new TaqMan HF183/BacR287 assay should provide more accurate estimations of human-derived fecal contaminants in ambient surface waters.
Gaudet, Daniel; Nilsson, Denise; Lohr, Tanner; Sheedy, Claudia
A competitive real-time (RT) immuno-polymerase chain reaction (iPCR) (RT-iPCR) assay was developed for the sensitive quantification of 17β-estradiol in water. Using a universal iPCR method and polyclonal antibodies, 17β-estradiol was accurately quantified at concentrations ranging from 1 pg mL(-1) to 10 µg mL(-1). The RT-iPCR assay's limit of detection was 0.7 pg mL(-1). The RT-iPCR assay provided an 800-fold increase in sensitivity as well as an expanded working range compared with the corresponding enzyme-linked immunosorbent assay. Assay cross-reactivity to estrone and estriol, two structurally related estrogens, was below 8%. Water samples spiked with 17β-estradiol were analyzed by RT-iPCR to determine the assay's potential as a rapid screen for the monitoring of manure-borne estrogens in the environment. The assay showed recoveries of 82, 102 and 103% for Milli-Q, tap, and irrigation water, respectively, without requiring sample extraction or concentration prior to analysis.
Green, Hyatt C.; Haugland, Richard A.; Varma, Manju; Millen, Hana T.; Borchardt, Mark A.; Field, Katharine G.; Walters, William A.; Knight, R.; Sivaganesan, Mano; Kelty, Catherine A.
Quantitative real-time PCR (qPCR) assays that target the human-associated HF183 bacterial cluster within members of the genus Bacteroides are among the most widely used methods for the characterization of human fecal pollution in ambient surface waters. In this study, we show that a current TaqMan HF183 qPCR assay (HF183/BFDrev) routinely forms nonspecific amplification products and introduce a modified TaqMan assay (HF183/BacR287) that alleviates this problem. The performance of each qPCR assay was compared in head-to-head experiments investigating limits of detection, analytical precision, predicted hybridization to 16S rRNA gene sequences from a reference database, and relative marker concentrations in fecal and sewage samples. The performance of the modified HF183/BacR287 assay is equal to or improves upon that of the original HF183/BFDrev assay. In addition, a qPCR chemistry designed to combat amplification inhibition and a multiplexed internal amplification control are included. In light of the expanding use of PCR-based methods that rely on the detection of extremely low concentrations of DNA template, such as qPCR and digital PCR, the new TaqMan HF183/BacR287 assay should provide more accurate estimations of human-derived fecal contaminants in ambient surface waters. PMID:24610857
...; Abbott Cardiovascular Systems, Inc. (Cardiovascular Device Manufacturing); Riverside County, CA An... of FTZ 153, requesting manufacturing authority on behalf of Abbott Cardiovascular Systems, Inc... production of cardiovascular devices including stents, catheters and guidewires. Components and materials...
Yu, Peng-bo; Li, Shen; Wei, Jing; Ma, Chang-an; Lu, Xiao-ling; DU, Shui-quan; Guan, Lu-yuan; Zheng, Yuan; Dong, Jian-hua; Ma, Chao-feng; Wang, Jing-jun
To compare the differences between the direct immuno-fluorescent assay (DFA) and real-time quantitative PCR in detecting the Hantavirus (HV) in rat lungs. From April to October in 2012, a total of 479 rats were caught by mouse-trap in residential or wild areas in Huxian, Jingyang, and Meixian of Shaanxi province, where haemorrhagic fever with renal syndrome (HFRS) was highly prevalent. The rats were dissected to take the two lungs, one was frozen and applied immuno-fluorescent assay to detect HV antigen while the other one was extracted its RNA and detected HV nucleic acid by real-time quantitative PCR. Then we compared the positive rate of the two methods. Out of the 479 rats, 105 were caught from residential areas and the other 374 were caught from wild areas. Among the 105 rats caught from residential areas, no HV were detected out neither by DFA nor by real-time quantitative PCR. Among the 374 wild rats, 13.1% (49/374) were detected HV positive by DFA and 14.7% (55/374) were detected HV positive by real-time quantitative PCR. The difference showed no statistical significance (χ(2) = 0.402, P = 0.526). When detecting each lung sample, the HV positive rate was 10.2% (49/479) under the detection by DFA while the HV positive rate was 11.5% (55/479) under the detection by real-time quantitative PCR. The difference had no statistical significance (χ(2) = 1.286, P = 0.257) and the consistency coefficient was 68.2% under the paired chi-square test analysis, which showed high consistency (u = 11.759, P < 0.05). The sensitivity of real-time quantitative PCR to detect HV was 77.6% (38/49) comparing with DFA as standard, and the specificity was 96.1% (413/430). Out of the 9 suspected HV positive sample detected by DFA, 6 were confirmed positive by real-time quantitative PCR and 3 were denied. Compared with the DFA, real-time quantitative PCR could also be used to detect the infection of HV in rats, and the result might be more stable.
Zhao, LiHong; Zhao, ShuPing
It is noted that more than 99 % of fluoroquinolone resistance in Neisseria gonorrhoeae (QRNG) specimens have been shown to have the mutation of Ser91/Phe in the gyrA gene. In order to detect QRNG isolates as quickly as possible, the real-time TaqMan quantitative PCR assay was established for detection of the point mutation of Ser91/Phe in gyrA gene. The standard curve was generated automatically on ABI Prism PE7500. The correlation coefficient (r) of the standard curve was -0.9984 (R(2) = 0.9968), indicating a quietly precise log-linear relationship between the concentration of target DNA and the Ct value. Presently, correlated, cultured antimicrobial susceptibility testing of N. gonorrhoeae isolates continues to be the gold standard method for the detection of antimicrobial resistance. Comparison to the correlated, cultured antimicrobial susceptibility testing, the sensitivity and specificity of the established TaqMan assay for the detection of the QRNG specimens were 100 and 99 %, respectively. The TaqMan assay also allows for rapid detection of QRNG isolates without complex laboratory techniques. Therefore, real-time TaqMan quantitative PCR assay is a rapid, simple, highly sensitive, highly specific, and easy-to-perform method for the detection of the QRNG specimens. It can be applied as a quick screening method for QRNG isolates to help clinical determination of optimal treatment prescription.
Stubbs, Samuel; Oura, Chris A L; Henstock, Mark; Bowden, Timothy R; King, Donald P; Tuppurainen, Eeva S M
Capripoxviruses, which are endemic in much of Africa and Asia, are the aetiological agents of economically devastating poxviral diseases in cattle, sheep and goats. The aim of this study was to validate a high-throughput real-time PCR assay for routine diagnostic use in a capripoxvirus reference laboratory. The performance of two previously published real-time PCR methods were compared using commercially available reagents including the amplification kits recommended in the original publication. Furthermore, both manual and robotic extraction methods used to prepare template nucleic acid were evaluated using samples collected from experimentally infected animals. The optimised assay had an analytical sensitivity of at least 63 target DNA copies per reaction, displayed a greater diagnostic sensitivity compared to conventional gel-based PCR, detected capripoxviruses isolated from outbreaks around the world and did not amplify DNA from related viruses in the genera Orthopoxvirus or Parapoxvirus. The high-throughput robotic DNA extraction procedure did not adversely affect the sensitivity of the assay compared to manual preparation of PCR templates. This laboratory-based assay provides a rapid and robust method to detect capripoxviruses following suspicion of disease in endemic or disease-free countries.
Pegels, Nicolette; González, Isabel; García, Teresa; Martín, Rosario
A ruminant-specific real-time PCR system was designed and applied for the detection of processed animal protein from ruminants in industrial feedstuffs. The assay includes a primer pair and a TaqMan probe selectively targeting mitochondrial 16S rRNA gene sequences from the ruminant group and another primer-probe set based on the eukaryotic nuclear 18S rRNA gene (positive amplification control). Both ruminant and eukaryotic PCR systems generated short PCR amplicons of 79 and 77 bp, respectively. To evaluate the suitability of the real-time PCR assay for the detection of banned by-products of ruminant origin, 126 feed samples subjected to rendering under current European legislation regulations were analyzed. The assay achieved 100% success in classifying the samples as positive or negative in terms of qualitative ruminant composition, with a detection limit of 0.1%. The quantitative ability of the assay is however restricted by variations in the composition and treatment of the feeds, which affect the amount and quality of amplifiable DNA.
Liu, Lihong; Xia, Hongyan; Everett, Helen; Sosan, Olubukola; Crooke, Helen; Meindl-Böhmer, Alexandra; Qiu, Hua-Ji; Moennig, Volker; Belák, Sándor; Widén, Frederik
Classical swine fever (CSF) is a highly contagious disease, causing severe economic losses in the pig industry worldwide. Vaccination of pigs with lapinized Chinese vaccines is still practised in some regions of the world, where the virus is enzootic, in order to prevent and control the disease. However, a single real-time assay that can detect all lapinized Chinese vaccines used widely, namely, Lapinized Philippines Coronel (LPC), Hog Cholera Lapinized virus (HCLV) and the Riems C-strain is still lacking. This study describes a real-time RT-PCR assay, targeting the N(pro) gene region, for specific detection of these lapinized vaccine strains. The assay is highly sensitive, with a detection limit of 10 genome copies per reaction for HCLV and Riems C-strain and highly specific, as more than 100 strains of wild type CSFV representing all major genotypes were not detected. The assay is also highly repeatable: the coefficient of variation of Ct values in three runs was 2.77% for the detection of 10 copies of the vaccine viral RNA. This study provides a potentially useful tool for specific detection of the lapinized Chinese vaccines, HCLV and C-strain, and the differentiation of these vaccines from wild type CSFV.
Černíková, Lenka; Vitásková, Eliška; Nagy, Alexander
Psittacine beak and feather disease (PBFD) is one of the most significant viral diseases in psittacine birds. The aim of the presented study was to develop a highly specific and sensitive TaqMan real-time PCR assay for universal detection of beak and feather disease virus (BFDV). Primers and a hydrolysis probe were selected on the highly conserved regions belonging to the ORF1 of the BFDV genome which were identified by aligning 814 genomic sequences downloaded from the GenBank database. The evaluation of the reaction parameters suggested a reaction efficiency of 97.1%, with consistent detection of 10(1) virus copies/μl of nucleic acid extract. The low values of standard deviation and coefficient of variation indicate a high degree of reproducibility and repeatability. The diagnostic applicability of the assay was proven on 36 BFDV positive and 107 negative specimens of psittacine origin representing 28 species. The assay showed a 100% ability to detect distinct genetic variants of the virus. Our data suggest that the presented TaqMan real-time PCR represents a specific, sensitive and reliable assay facilitating the molecular detection of BFDV.
Leon, Olivier; Corrand, Léni; Bich, Tran Ngoc; Le Minor, Odile; Lemaire, Mylène; Guérin, Jean-Luc
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.
Xia, Xiaoming; Yu, Yongxin; Hu, Linghao; Weidmann, Manfred; Pan, Yingjie; Yan, Shuling; Wang, Yongjie
Infectious hypodermal and hematopoietic necrosis virus (IHHNV) causes mortality or runt deformity syndrome in penaeid shrimps and is responsible for significant economic losses in the shrimp aquaculture industry. Here, we describe a novel real-time isothermal recombinase polymerase amplification (RPA) assay developed for IHHNV detection. Using IHHNV plasmid standards and DNA samples from a variety of organisms, we evaluated the ability of the IHHNV-RPA assay to detect IHHNV based on analysis of its sensitivity, specificity, rapidity, and reproducibility. Probit analysis of eight independent experimental replicates indicated satisfactory performance of the RPA assay, which is sufficiently sensitive to detect as few as 4 copies of the IHHNV genome within 7 min at 39 °C with 95 % reliability. Therefore, this rapid RPA method has great potential for applications, either in field use or as a point of care diagnostic technique.
Marshall, R.; Chernesky, M.; Jang, D.; Hook, E. W.; Cartwright, C. P.; Howell-Adams, B.; Ho, S.; Welk, J.; Lai-Zhang, J.; Brashear, J.; Diedrich, B.; Otis, K.; Webb, E.; Robinson, J.; Yu, H.
We evaluated a new real-time PCR-based prototype assay for the detection of Chlamydia trachomatis and Neisseria gonorrhoeae developed by Abbott Molecular Inc. This assay is designed to be performed on an Abbott m2000 real-time instrument system, which consists of an m2000sp instrument for sample preparation and an m2000rt instrument for real-time PCR amplification and detection. The limit of detection of this prototype assay was determined to be 20 copies of target DNA for both C. trachomatis and N. gonorrhoeae, using serially diluted linearized plasmids. No cross-reactivity could be detected when 55 nongonococcal Neisseria isolates and 3 non-C. trachomatis Chlamydia isolates were tested at 1 million genome equivalents per reaction. Concordance with the Roche Amplicor, BDProbeTec ET, and Gen-Probe APTIMA Combo 2 tests was assessed using unlinked/deidentified surplus clinical specimens previously analyzed with these tests. For C. trachomatis, concordance for positive results ranged from 93.7% to 100%, while concordance for negative results ranged from 98.2% to 100%. For N. gonorrhoeae, concordance for positive and negative results ranged from 91.4% to 100% and 99.3% to 100%, respectively. A workflow analysis of the prototype assay was conducted to obtain information on throughput under laboratory conditions. At 48 samples/run, the time to first result for both C. trachomatis and N. gonorrhoeae was 4.5 h. A total of 135 patient specimens could be analyzed in 8.9 h, with 75 min of hands-on time. This study demonstrated the technical and clinical feasibility of the new Abbott real-time PCR C. trachomatis/N. gonorrhoeae assay. PMID:17202273
42. Peaks of Otter, Abbott Lake. View across lake to peaks of Outter Lodge, completed in 1964. Construction of the lake got underway in 1964. Looking east-northeast. - Blue Ridge Parkway, Between Shenandoah National Park & Great Smoky Mountains, Asheville, Buncombe County, NC
New Jersey's urban--or "Abbott"--schools have improved at the preschool and elementary school level, but lag when it comes to middle and high school performance. These are the key findings of an Abbott Indicators Project report entitled, "The Abbott Districts in 2005-06: Progress and Challenges." The report was prepared by…
Hoffman, Shari Cole
This article profiles Grace Abbott, one of the earlier 20th century American women leaders in Progressivism. Abbott's heritage influenced her lifetime commitment to social improvement. She was born on November 17, 1878 in Grand Island, Nebraska into a family of activists. Her Quaker mother, Elizabeth Griffin Abbott, came from an abolitionist…
Hoffman, Shari Cole
This article profiles Grace Abbott, one of the earlier 20th century American women leaders in Progressivism. Abbott's heritage influenced her lifetime commitment to social improvement. She was born on November 17, 1878 in Grand Island, Nebraska into a family of activists. Her Quaker mother, Elizabeth Griffin Abbott, came from an abolitionist…
Iraola, Gregorio; Pérez, Ruben; Betancor, Laura; Marandino, Ana; Morsella, Claudia; Méndez, Alejandra; Paolicchi, Fernando; Piccirillo, Alessandra; Tomás, Gonzalo; Velilla, Alejandra; Calleros, Lucía
Campylobacter fetus is a pathogen of major concern for animal and human health. The species shows a great intraspecific variation, with three subspecies: C. fetus subsp. fetus, C. fetus subsp. venerealis, and C. fetus subsp. testudinum. Campylobacter fetus fetus affects a broad range of hosts and induces abortion in sheep and cows. Campylobacter fetus venerealis is restricted to cattle and causes the endemic disease bovine genital campylobacteriosis, which triggers reproductive problems and is responsible for major economic losses. Campylobacter fetus testudinum has been proposed recently based on genetically divergent strains isolated from reptiles and humans. Both C. fetus fetus and C. fetus testudinum are opportunistic pathogens for immune-compromised humans. Biochemical tests remain as the gold standard for identifying C. fetus but the fastidious growing requirements and the lack of reliability and reproducibility of some biochemical tests motivated the development of molecular diagnostic tools. These methods have been successfully tested on bovine isolates but fail to detect some genetically divergent strains isolated from other hosts. The aim of the present study was to develop a highly specific molecular assay to identify and quantify C. fetus strains. We developed a highly sensitive real-time PCR assay that targets a unique region of the 16S rRNA gene. This assay successfully detected all C. fetus strains, including those that were negative for the cstA gene-based assay used as a standard for molecular C. fetus identification. The assay showed high specificity and absence of cross-reactivity with other bacterial species. The analytical testing of the assay was determined using a standard curve. The assay demonstrated a wide dynamic range between 10(2) and 107 genome copies per reaction, and a good reproducibility with small intra- and inter-assay variability. The possibility to characterize samples in a rapid, sensitive and reproducible way makes this assay
Deng, Jingjing; Yu, Ping; Wang, Yuexiang; Mao, Lanqun
This study demonstrates a novel ratiometric fluorescent method for real-time alkaline phosphatase (ALP) activity assay with stimulus responsive infinite coordination polymer (ICP) nanoparticles as the probe. The ICP nanoparticles used in this study are composed of two components; one is the supramolecular ICP network formed with guanine monophosphate (GMP) as the ligand and Tb(3+) as the central metal ion, and the other is a fluorescent dye, i.e., 7-amino-4-methyl coumarin (coumarin) encapsulated into the ICP network. Upon being excited at 315 nm, the ICP network itself emits green fluorescence at 552 nm. Coumarin dye encapsulated in the ICP network emits weak fluorescence at 450 nm upon excitation at the same wavelength (315 nm), and this fluorescence emission becomes strong when the encapsulated dye is released from the network into the solution phase. Hence, we develop a ratiometric fluorescent assay based on the ALP-induced destruction of the supramolecular ICP network and the release of coumarin. This mechanism can be used for real-time ratiometric fluorescent monitoring of ALP activity by continuously measuring the ratio of fluorescent intensity at the wavelength of 552 nm (F552) to that at 450 nm (F450) (F552/F450) in the time-dependent fluorescent spectra of the coumarin@Tb-GMP suspension containing ALP with different activities. Under the experimental conditions employed here, the F552/F450 value is linear with the ALP activity within a range from 0.025 U/mL to 0.2 U/mL. The detection limit is down to 0.010 U/mL (S/N = 3). Moreover, the assay developed here is employed for ALP inhibitor evaluation. This study offers a simple yet sensitive method for real-time ALP activity assay.
Lapierre, Pascal; Huletsky, Ann; Fortin, Véronique; Picard, François J.; Roy, Paul H.; Ouellette, Marc; Bergeron, Michel G.
Resistance to fluoroquinolones among clinical isolates of Staphylococcus aureus has become a clinical problem. Therefore, a rapid method to identify S. aureus and its susceptibility to fluoroquinolones could provide clinicians with a useful tool for the appropriate use of these antimicrobial agents in the health care settings. In this study, we developed a rapid real-time PCR assay for the detection of S. aureus and mutations at codons Ser-80 and Glu-84 of the grlA gene encoding the DNA topoisomerase IV, which are associated with decreased susceptibility to fluoroquinolones. The detection limit of the assay was 10 genome copies per reaction. The PCR assay was negative with DNA from all 26 non-S. aureus bacterial species tested. A total of 85 S. aureus isolates with various levels of fluoroquinolone resistance was tested with the PCR assay. The PCR assay correctly identified 100% of the S. aureus isolates tested compared to conventional culture methods. The correlation between the MICs of ciprofloxacin, levofloxacin, and gatifloxacin and the PCR results was 98.8%. The total time required for the identification of S. aureus and determination of its susceptibility to fluoroquinolones was about 45 min, including DNA extraction. This new rapid PCR assay represents a powerful method for the detection of S. aureus and its susceptibility to fluoroquinolones. PMID:12843071
Zhu, Tong; Zhao, Guimin; Shen, Furao; Hou Peili; Wang, Hongmei; Li, Jie; He, Hongbin
The bovine enterovirus (BEV) is a pathogen found the digestive tracts of cattle. Recently, the BEV was discovered in cattle in a province in China. A rapid and effective detection method for the BEV is essential. An assay was carried out using two specific primers designed to amplify a highly conserved sequence of the 3D gene. A recombinant plasmid containing the target gene 3D was constructed as a standard control. The limit of detection of the reaction was 7.13 x 10(1) plasmid copies/μL of initial templates, which was tenfold more sensitive than the conventional reverse-transcription-polymerase chain reaction (RT-PCR). Moreover, the assay was highly specific because all negative controls and other viruses of clinical relevance did not develop positive results. Assay performance on field samples was evaluated on 44 (41 diarrhea and 3 aerosol) samples and compared with the conventional RT-PCR assay. Sixteen diarrhea samples were positive (16/41, 39. 02%) and 3 aerosol samples were positive (3/3, 100%). Preliminary results for clinical detection showed that the SYBR Green I real-time PCR assay was highly sensitive, specific and reproducible. The robustness and high-throughput performance of the developed assay make it a powerful tool in diagnostic applications for epidemics and in BEV research.
Caviglia, C; Zór, K; Canepa, S; Carminati, M; Larsen, L B; Raiteri, R; Andresen, T L; Heiskanen, A; Emnéus, J
We investigated the combined effect of the initial cell density (12,500, 35,000, 75,000, and 100,000 cells cm(-2)) and concentration of the anti-cancer drug doxorubicin on HeLa cells by performing time-dependent cytotoxicity assays using real-time electrochemical impedance spectroscopy. A correlation between the rate of cell death and the initial cell seeding density was found at 2.5 μM doxorubicin concentration, whereas this was not observed at 5 or 100 μM. By sensing the changes in the cell-substrate interaction using impedance spectroscopy under static conditions, the onset of cytotoxicity was observed 5 h earlier than when using a standard colorimetric end-point assay (MTS) which measures changes in the mitochondrial metabolism. Furthermore, with the MTS assay no cytotoxicity was observed after 15 h of incubation with 2.5 μM doxorubicin, whereas the impedance showed at this time point cell viability that was below 25%. These results indicate that impedance detection reveals cytotoxic events undetectable when using the MTS assay, highlighting the importance of combining impedance detection with traditional drug toxicity assays towards a more in depth understanding of the effect of anti-cancer drugs on in vitro assays. Moreover, the detection of doxorubicin induced toxicity determined with impedance under static conditions proved to be 6 times faster than in perfusion culture.
Ji, Xiaolin; Wang, Qi; Gao, Yulong; Wang, Yongqiang; Qin, Liting; Qi, Xiaole; Gao, Honglei; Wang, Xiaomei
Recently, there has been a growing body of evidence showing that cellular microRNAs (miRNAs) are involved in virus-host interactions. Numerous studies have focused on analyses of the expression profiles of cellular miRNAs, but the expression patterns of Dicer, which is responsible for the generation of miRNAs, have only rarely been explored in Gallus gallus. We developed a duplex realtime reverse transcriptase polymerase chain reaction (RTPCR) assay for the relative quantification of the mRNAs of Dicer and beta-actin in G. gallus. To apply this method, the expression of Dicer in avian cells after infection with avian leukosis virus subgroup J (ALV-J) was detected using our established duplex real-time RT-PCR. The duplex realtime RT-PCR assay is sufficiently sensitive, specific, accurate, reproducible, and cost-effective for the detection of Dicer in G. gallus. Furthermore, this study, for the first time, demonstrated that ALV-J can induce differential expression of Dicer mRNA in the ALV-J-infected cells.
Sandeu, Maurice Marcel; Moussiliou, Azizath; Moiroux, Nicolas; Padonou, Gilles G.; Massougbodji, Achille; Corbel, Vincent; Tuikue Ndam, Nicaise
Background An accurate method for detecting malaria parasites in the mosquito’s vector remains an essential component in the vector control. The Enzyme linked immunosorbent assay specific for circumsporozoite protein (ELISA-CSP) is the gold standard method for the detection of malaria parasites in the vector even if it presents some limitations. Here, we optimized multiplex real-time PCR assays to accurately detect minor populations in mixed infection with multiple Plasmodium species in the African malaria vectors Anopheles gambiae and Anopheles funestus. Methods Complementary TaqMan-based real-time PCR assays that detect Plasmodium species using specific primers and probes were first evaluated on artificial mixtures of different targets inserted in plasmid constructs. The assays were further validated in comparison with the ELISA-CSP on 200 field caught Anopheles gambiae and Anopheles funestus mosquitoes collected in two localities in southern Benin. Results The validation of the duplex real-time PCR assays on the plasmid mixtures demonstrated robust specificity and sensitivity for detecting distinct targets. Using a panel of mosquito specimen, the real-time PCR showed a relatively high sensitivity (88.6%) and specificity (98%), compared to ELISA-CSP as the referent standard. The agreement between both methods was “excellent” (κ = 0.8, P<0.05). The relative quantification of Plasmodium DNA between the two Anopheles species analyzed showed no significant difference (P = 0, 2). All infected mosquito samples contained Plasmodium falciparum DNA and mixed infections with P. malariae and/or P. ovale were observed in 18.6% and 13.6% of An. gambiae and An. funestus respectively. Plasmodium vivax was found in none of the mosquito samples analyzed. Conclusion This study presents an optimized method for detecting the four Plasmodium species in the African malaria vectors. The study highlights substantial discordance with traditional ELISA-CSP pointing out the
Lucas, Tamara; Villegas, Ana Maria
In 1999-2000, over one-third of all students in the 30 Abbott districts spoke a native language other than English, and more than one-tenth were considered limited English proficient (LEP). The proportions of LEP students varied considerably across the districts, but they comprised between 5% and 29% of total enrollments in 18 of the districts.…
Yang, Yang; Qin, Xiaodong; Zhang, Xiangle; Zhao, Zhixun; Zhang, Wei; Zhu, Xueliang; Cong, Guozheng; Li, Yanmin; Zhang, Zhidong
Goatpox virus (GTPV) and sheeppox virus (SPPV), which belong to the Capripoxvirus (CaPV), are economically important pathogens of small ruminants. Therefore, a sensitive, specific and rapid diagnostic assay for detection of GTPV and SPPV is necessary to accurately and promptly control these diseases. Recombinase polymerase amplification (RPA) assays combined with a real-time fluorescent detection (real-time RPA assay) and lateral flow dipstick (RPA LFD assay) were developed targeting the CaPV G-protein-coupled chemokine receptor (GPCR) gene, respectively. The sensitivity of both CaPV real-time RPA assay and CaPV RPA LFD assay were 3 × 10(2) copies per reaction within 20 min at 38 °C. Both assays were highly specific for CaPV, with no cross-reactions with peste des petits ruminants virus, foot-and-mouth disease virus and Orf virus. The evaluation of the performance of these two assays with clinical sample (n = 107) showed that the CaPV real-time RPA assay and CaPV RPA LFD assay were able to specially detect SPPV or GTPV present in samples of ovine in liver, lung, kidney, spleen, skin and blood. This study provided a highly time-efficient and simple alternative for rapid detection of GTPV and SPPV.
Palle-Reisch, Monika; Hochegger, Rupert; Štumr, Stepan; Korycanova, Kveta; Cichna-Markl, Margit
The study compares the applicability of two commercial mustard ELISA kits (Mustard ELISA Kit-specific and Mustard ELISA Kit-total) and three in-house developed real-time PCR assays (singleplex assay for white mustard, singleplex assay for black/brown mustard and duplex assay for the detection of white, black and brown mustard). Analyses of raw and brewed model sausages containing white and black/brown mustard in the range from 1 to 50 ppm indicate that both ELISAs and the three real-time PCR assays allow the detection of traces of mustard in raw and in brewed sausages. The ELISAs were found to be more sensitive than the real-time PCR assays. When the ELISAs and real-time PCR assays were applied to the analysis of 15 commercial foodstuffs differing in their labelling concerning mustard, in one sample mustard was detected with both ELISAs and the three real-time PCR assays although mustard was not indicated on the food ingredient list. Copyright © 2014 Elsevier Ltd. All rights reserved.
Vargas, Diana Y.; Kramer, Fred Russell; Tyagi, Sanjay; Marras, Salvatore A. E.
We describe the use of “SuperSelective” primers that enable the detection and quantitation of somatic mutations whose presence relates to cancer diagnosis, prognosis, and therapy, in real-time PCR assays that can potentially analyze rare DNA fragments present in blood samples (liquid biopsies). The design of these deoxyribonucleotide primers incorporates both a relatively long “5' anchor sequence” that hybridizes strongly to target DNA fragments, and a very short, physically and functionally separate, “3' foot sequence” that is perfectly complementary to the mutant target sequence, but mismatches the wild-type sequence. As few as ten mutant fragments can reliably be detected in the presence of 1,000,000 wild-type fragments, even when the difference between the mutant and the wild type is only a single nucleotide polymorphism. Multiplex PCR assays employing a set of SuperSelective primers, and a corresponding set of differently colored molecular beacon probes, can be used in situations where the different mutations, though occurring in different cells, are located in the same codon. These non-symmetric real-time multiplex PCR assays contain limited concentrations of each SuperSelective primer, thereby enabling the simultaneous determination of each mutation’s abundance by comparing its threshold value to the threshold value of a reference gene present in the sample. PMID:27244445
Boddicker, Jennifer D.; Rota, Paul A.; Kreman, Trisha; Wangeman, Andrea; Lowe, Louis; Hummel, Kimberly B.; Thompson, Robert; Bellini, William J.; Pentella, Michael; DesJardin, Lucy E.
The mumps virus is a negative-strand RNA virus in the family Paramyxoviridae. Mumps infection results in an acute illness with symptoms including fever, headache, and myalgia, followed by swelling of the salivary glands. Complications of mumps can include meningitis, deafness, pancreatitis, orchitis, and first-trimester abortion. Laboratory confirmation of mumps infection can be made by the detection of immunoglobulin M-specific antibodies to mumps virus in acute-phase serum samples, the isolation of mumps virus in cell culture, or by detection of the RNA of the mumps virus by reverse transcription (RT)-PCR. We developed and validated a multiplex real-time RT-PCR assay for rapid mumps diagnosis in a clinical setting. This assay used oligonucleotide primers and a TaqMan probe targeting the mumps SH gene, as well as primers and a probe that targeted the human RNase P gene to assess the presence of PCR inhibitors and as a measure of specimen quality. The test was specific, since it did not amplify a product from near-neighbor viruses, as well as sensitive and accurate. Real-time RT-PCR results showed 100% correlation with results from viral culture, the gold standard for mumps diagnostic testing. Assay efficiency was over 90% and displayed good precision after performing inter- and intraassay replicates. Thus, we have developed and validated a molecular method for rapidly diagnosing mumps infection that may be used to complement existing techniques. PMID:17652480
Blanco-Lobo, P; González-Galán, V; García-Quintanilla, M; Valencia, R; Cazalla, A; Martín, C; Alonso, I; Pérez-Romero, P; Cisneros, J M; Aznar, J; McConnell, M J
Real-time polymerase chain reaction (PCR)-based approaches have not been assessed in terms of their ability to detect patients colonized by Acinetobacter baumannii during active surveillance. This prospective, double-blind study demonstrated that a real-time PCR assay had high sensitivity (100%) and specificity (91.2%) compared with conventional culture for detecting A. baumannii in 397 active surveillance samples, and provided results within 3h. Receiver-operator curve analyses demonstrated that the technique has diagnostic accuracy of 97.7% (95% confidence interval 96.0-99.3%). This method could facilitate the rapid implementation of infection control measures for preventing the transmission of A. baumannii.
Pantchev, Alexandra; Sting, Reinhard; Bauerfeind, Rolf; Tyczka, Judith; Sachse, Konrad
The aim of the present study was to analyse the occurrence of chlamydiae in several mammalian host species. Clinical samples that previously tested positive in a Chlamydiaceae-specific real-time PCR were retested using six species-specific real-time PCR assays to identify the chlamydial species involved. Chlamydophila (Cp.) abortus was the agent most frequently found in cattle, sheep, horses, goats, and pigs. Detection in cattle of Cp. psittaci (11% of samples) and Chlamydia (C.) suis (9%), as well as Cp. psittaci in a goat sample was somewhat unexpected. DNA of two different chlamydiae was identified in 56 (12.7%) of 440 samples tested. Cp. felis was the predominant species found in cats, while in guinea pigs and rabbits only Cp. caviae was detected. Interestingly, the latter two pathogens were also identified in samples from dogs. The data show that mixed chlamydial infections are not rare and suggest an extended host range of individual species.
Gordon, Katrina V.; Vickery, Michael C.; DePaola, Angelo; Staley, Christopher; Harwood, Valerie J.
Vibrio vulnificus is an autochthonous estuarine bacterium and a pathogen that is frequently transmitted via raw shellfish. Septicemia can occur within 24 h; however, isolation and confirmation from water and oysters require days. Real-time PCR assays were developed to detect and differentiate two 16S rRNA variants, types A and B, which were previously associated with environmental sources and clinical fatalities, respectively. Both assays could detect 102 to 103 V. vulnificus total cells in seeded estuarine water and in oyster homogenates. PCR assays on 11 reference V. vulnificus strains and 22 nontarget species gave expected results (type A or B for V. vulnificus and negative for nontarget species). The relationship between cell number and cycle threshold for the assays was linear (R2 = >0.93). The type A/B ratio of Florida clinical isolates was compared to that of isolates from oysters harvested in Florida waters. This ratio was 19:17 in clinical isolates and 5:8 (n = 26) in oysters harvested from restricted sites with poor water quality but was 10:1 (n = 22) in oysters from permitted sites with good water quality. A substantial percentage of isolates from oysters (19.4%) were type AB (both primer sets amplified), but no isolates from overlying waters were type AB. The real-time PCR assays were sensitive, specific, and quantitative in water samples and could also differentiate the strains in oysters without requiring isolation of V. vulnificus and may therefore be useful for rapid detection of the pathogen in shellfish and water, as well as further investigation of its population dynamics. PMID:18245234
Haque, Rashidul; Kabir, Mamun; Noor, Zannatun; Rahman, S M Mazidur; Mondal, Dinesh; Alam, Faisal; Rahman, Intekhab; Al Mahmood, Abdullh; Ahmed, Nooruddin; Petri, William A
The noninvasive diagnosis of amebic liver abscess is challenging, as most patients at the time of diagnosis do not have a concurrent intestinal infection with Entamoeba histolytica. Fecal testing for E. histolytica parasite antigen or DNA is negative in most patients. A real-time PCR assay was evaluated for detection of E. histolytica DNA in blood, urine, and saliva samples from amebic liver abscess as well as amebic colitis patients in Bangladesh. A total of 98 amebic liver abscess and 28 amebic colitis patients and 43 control subjects were examined. The real-time PCR assay detected E. histolytica DNA in 49%, 77%, and 69% of blood, urine, and saliva specimens from the amebic liver abscess patients. For amebic colitis the sensitivity of the real-time PCR assay for detection of E. histolytica DNA in blood, urine, and saliva was 36%, 61%, and 64%, respectively. All blood, urine, and saliva samples from control subjects were negative by the real-time PCR assay for E. histolytica DNA. When the real-time PCR assay results of the urine and saliva specimens were taken together (positive either in urine or saliva), the real-time PCR assay was 97% and 89% sensitive for detection of E. histolytica DNA in liver abscess and intestinal infection, respectively. We conclude that the detection of E. histolytica DNA in saliva and urine could be used as a diagnostic tool for amebic liver abscess.
Chaturvedi, Sudha; Rudd, Robert J; Davis, April; Victor, Tanya R; Li, Xiaojiang; Appler, Kim A; Rajkumar, Sunanda S; Chaturvedi, Vishnu
Geomyces destructans is the etiologic agent of bat geomycosis, commonly referred to as white nose syndrome (WNS). This infection has caused severe morbidity and mortality in little brown bats (Myotis lucifugus) and has also spread to other bat species with significant decline in the populations. Currently, G. destructans infection is identified by culture, ITS-PCR, and histopathology. We hypothesized that a real-time PCR assay would considerably improve detection of G. destructans in bats. The 100 bp sequence of the Alpha-L-Rhamnosidase gene was validated as a target for real-time PCR. The assay sensitivity was determined from serial dilution of DNA extracted from G. destructans conidia (5 × 10(-1)-5 × 10(7)), and the specificity was tested using DNA from 30 closely and distantly related fungi and 5 common bacterial pathogens. The real-time PCR assay was highly sensitive with detection limit of two G. destructans conidia per reaction at 40 PCR cycles. The assay was also highly specific as none of the other fungal or bacterial DNA cross-reacted in the real-time PCR assay. One hundred and forty-seven bat tissue samples, suspected of infection with G. destructans, were used to compare the real-time PCR assay to other methods employed for the detection of G. destructans. Real-time PCR was highly sensitive with 80 of 147 (55%) samples testing positive for G. destructans DNA. In comparison, histopathology examination revealed 64/147 (44%) positive samples. The internal transcribed spacer (ITS)-PCR yielded positive amplicon for G. destructans from 37 tissue samples (25%). The least sensitive assay was the fungal culture with only 17 tissue samples (12%) yielding G. destructans in culture. The data suggested that the real-time PCR assay is highly promising for rapid, sensitive, and specific identification of G. destructans. Further trials and inter-laboratory comparisons of this novel assay are recommended to improve the diagnosis of bat geomycosis.
Qian, Zhaosheng; Chai, Lujing; Tang, Cong; Huang, Yuanyuan; Chen, Jianrong; Feng, Hui
A convenient, reliable, and highly sensitive real-time assay for alkaline phosphatase (ALP) activity in the continuous and recyclable way is established on the basis of aggregation and disaggregation of carbon quantum dots (CQDs) through the competitive assay approach. CQDs and adenosine triphosphate (ATP) were used as the fluorescent indicator and substrate for ALP activity assessment, respectively. Richness of carboxyl groups on the surface of CQDs enables their severe aggregation triggered by cerium ions, which results in effective fluorescence quenching. Under the catalytic hydrolysis of ALP, ATP can be rapidly transformed to phosphate ions. Stronger affinity of phosphate ions to cerium ions than carboxyl groups is taken advantage of to achieve fluorescence recovery induced by redispersion of CQDs in the presence of ALP and ATP. Quantitative evaluation of ALP activity in a broad range from 4.6 to 383.3 U/L with the detection limit of 1.4 U/L can be realized in this way, which endows the assay with high enough sensitivity for practical detection in human serum. The assay can be used in a recyclable way for more than three times since the generated product CePO4 as a precipitate can be easily removed from the standard assay system. This strategy broadens the sensing application of fluorescent CQDs with excellent biocompatibility and provides an example based on disaggregation in optical probe development.
Grant, Rebecca J; Banyard, Ashley C; Barrett, Tom; Saliki, Jeremiah T; Romero, Carlos H
Real-time RT-PCR (rtRT-PCR) assays for identifying and differentiating infections caused by dolphin morbillivirus (DMV) and porpoise morbillivirus (PMV) were developed by targeting the hypervariable C-terminal domain of the nucleocapsid (N) gene. Total DMV and PMV RNA extracted from infected Vero cells expressing the canine signaling lymphocyte-activation molecule (SLAM) produced positive cycle threshold (C(T)) values after the 17th and 25th cycles, respectively. The assays were then validated using infected cetacean tissue RNA. The assays were specific for either DMV or PMV and did not cross-react with canine distemper virus (CDV), phocid distemper virus (PDV), rinderpest virus (RPV), peste des petits ruminants virus (PPRV) and measles virus (MV). The glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene was targeted as control for RNA quality, and a consensus GAPDH probe that reacted with 11 different marine mammal species, generating positive C(T) values ranging from the 21st to the 37th cycle was used. The rtRT-PCR assays have advantages over conventional assays in that they are rapid, easier to scale up, and are less prone to cross-contamination and have improved the limit of detection and specificity.
Zang, Yang; Du, Dongchuan; Ge, Peng; Xu, Yongqing; Liu, Xintao; Zhang, Yan; Su, Weiheng; Kiseleva, Irina; Rudenko, Larisa; Xu, Fei; Kong, Wei; Jiang, Chunlai
Traditionally, infectivity of a trivalent live attenuated influenza vaccines (LAIVs) is titrated by determining the 50% egg infectious dose assay (EID50) or plaque forming units (PFU), which requires specific monoclonal antibodies to neutralize 2 strains while estimating the titer of the non-neutralized strain. Compared to this time-consuming, laborious, subjective and variable process, reverse transcription-quantitative real-time PCR (RT-qPCR) technology has advantages of rapidity, sensitivity, reproducibility and reduced contamination, thus has been applied widely for detecting pathogens and measuring viral titers. In this study, the critical harvest time was determined to be 18 h post-infection (hpi) for type A influenza and 12 hpi for type B influenza, but no significant difference between titers at 12 hpi and 18 hpi for the type B strain was observed. In conclusion, trivalent LAIVs can be titrated simultaneously within 24 h by this one-step RT-qPCR assay, which yielded titers comparable to those obtained by the traditional EID50 assay. Therefore, the RT-qPCR assay may be used as a highly specific, sensitive, precise and rapid alternative to the EID50 assay for titering LAIVs. PMID:25483696
Nowrouzian, Forough L; Adlerberth, Ingegerd; Wold, Agnes E
Molecular biological methods using real-time polymerase chain reaction (RT-PCR) for detection of bacterial and viral genes in different environments have been developed into assays from different commercial sources. Applied Biosystems include and support two applications with their TaqMan instrument: the "Plus/Minus" and the "Allelic Discrimination" assays. These approaches are RT-PCR based, use short primers and fluorescent-labeled TaqMan probes and include three processes: a pre-read run, a PCR-amplification run, and a post-read run. In the "Plus/Minus" assay, samples and controls (distilled water) are loaded into the instrument, which calculates a positive or a negative outcome based on differences in signals between samples and the controls. When testing the "Plus/Minus" assay for detection of usp genes encoding a uropathogenic specific protein in Escherichia coli, an inordinately high proportion of false-positive signals was observed. This was shown to be due to a serious methodological deficiency. Our observations indicate that an adequate no-template control closely matching the target samples in all aspects, including amount of DNA, is required to establish a correct threshold in the pre-read run that forms the basis for further calculations in the post-read run of the "Plus/Minus" assay.
Wang, Heng; Nattanmai, Seela; Kramer, Laura D.; Bernard, Kristen A.; Tavakoli, Norma P.
A duplex TaqMan real-time RT-PCR assay was developed for the detection of California (CAL) serogroup viruses and Cache Valley virus (CVV), for use in human surveillance. The targets selected for the assay were the sequences encoding the nucleocapsid protein of CAL and the G1 glycoprotein of CVV. Conserved regions were selected by aligning genetic sequences from various strains available in the GenBank database. Primers and probes were selected in conserved regions. The assay sensitivity was 75 gene copies (gc)/reaction for CAL serogroup viruses and 30 gc/reaction for CVV. The performance of the assay was linear over at least 6 log10 gc. The assay was specific, given that it did not cross-react with a variety of pathogens. It did however, detect 11 viruses within the CAL serogroup, and 12 CVV isolates. The use of an internal control ensured that possible inefficiency in nucleic acid extraction or PCR inhibition would be detected. PMID:19748425
Pedersen, J.; Killian, M.L.; Hines, N.; Senne, D.; Panigrahy, B.; Ip, H.S.; Spackman, Erica
This report describes the validation of an avian influenza virus (AIV) H7 subtype-specific real-time reverse transcriptasePCR (rRT-PCR) assay developed at the Southeast Poultry Research Laboratory (SEPRL) for the detection of H7 AI in North and South American wild aquatic birds and poultry. The validation was a collaborative effort by the SEPRL and the National Veterinary Services Laboratories. The 2008 H7 rRT-PCR assay detects 101 50% embryo infectious doses per reaction, or 103104 copies of transcribed H7 RNA. Diagnostic sensitivity and specificity were estimated to be 97.5% and 82.4%, respectively; the assay was shown to be specific for H7 AI when tested with >270 wild birds and poultry viruses. Following validation, the 2008 H7 rRT-PCR procedure was adopted as an official U.S. Department of Agriculture procedure for the detection of H7 AIV. The 2008 H7 assay replaced the previously used (2002) assay, which does not detect H7 viruses currently circulating in wild birds in North and South America. ?? 2010 American Association of Avian Pathologists.
Xu, Shushen; Green, Michael; Kingsley, Laurence; Webber, Steven; Rowe, David
Monitoring the load of Epstein-Barr virus (EBV) in the peripheral blood by quantitative PCR has been accepted as a useful tool for predicting the onset of EBV related diseases, confirming an EBV disease diagnosis and following the response to treatment interventions. In the present study, the use of a realtime polymerase chain reaction (rt-PCR) assay developed for unpurified cell preparations was examined and the results of the realtime assay were compared to an EBV quantitative-competitive PCR assay (QC-PCR). Both assays use the same target sequence and the same method for determining the standard value for the copy number of EBV genomes present. A comparison of 572 PCR results reveals that the realtime assay gave 5-10-fold higher values than the QC-PCR. Fifty-one results (8.9%) were discordant between the two sets of data. The most commonly encountered discordant result was detection of low amounts of EBV DNA by the rt-PCR assay that were not detected in specimens by QC-PCR. The two assays had a high degree of correlation across the range of load detection allowing clinically relevant threshold values determined in the QC-PCR assay to be inferred for the rt-PCR assay. External normalization of the rt-PCR assay was determined to be an important tool for monitoring the quality and/or quantity of human DNA in the starting material. rt-PCR assays with unpurified cell lysates compare favorably with quantitative-competitive assays and when normalized offer real advantages in specimen preparation, assay manipulations and reproducibility over both quantitative-competitive assays and realtime assays that require purified nucleic acid inputs.
Martinot-Peignoux, Michelle; Khiri, Hacène; Leclere, Laurence; Maylin, Sarah; Marcellin, Patrick; Halfon, Philippe
Early viral monitoring is essential for the management of treatment outcome in patients with chronic hepatitis C. A variety of commercially available assays are now available to quantify HCV-RNA in routine clinical practice. Compare the clinical results of 3 commercially available assays to evaluate the positive predictive value (PPV) and the negative predictive value (NPV) of rapid virological response (RVR) at week 4 and early virological response (EVR) at week 12. 287 patients treated with standard care regimen combination therapy were studied. HCV-RNA values measured at baseline, week 4, week 12 with VERSANT HCV 3.0 Assay (bDNA), and VERSANT HCV-RNA Qualitative Assay (TMA) (bDNA/TMA); COBAS Ampliprep/COBAS/TaqMan (CAP/CTM) and Abbott m2000sp extraction/m2000rt amplification system (ART). RVR was defined as undetectable serum HCV-RNA and EVR as a > OR =2 log decline in baseline viral load (BLV). Median (range) BVLs were: 5.585(2.585-6.816), 5.189(2.792-7.747) and 4.804(2.380-6.580) log(10)IU/ml, with bDNA/TMA, CAP/CTM and ART, respectively (p<0.01); RVR was observed in 22%, 30% and 27% of the patients and PPVs were 97%, 91% and 94% with bDNA/TMA, CAP/CTM and ART, respectively (p=0.317). EVR was observed in 76%, 73% and 67% of the patients and NPVs were 93%, 83% and 79% with bDNA/TMA, CAP/CTM and ART, respectively (p=0.09). Treatment monitoring should include both detection of serum HCV-RNA at week 4 to predict SVR and at week 12 to predict non-SVR. The value of all 3 assays was similar for evaluating RVR or EVR. Because of viral load discrepancies the same assay should be used throughout patient treatment follow-up.
Whitehouse, Chris A.; Chase, Kitty; Embers, Monica E.; Kulesh, David A.; Ladner, Jason T.; Palacios, Gustavo F.; Minogue, Timothy D.
Background Moraxella macacae is a recently described bacterial pathogen that causes epistaxis or so-called bloody nose syndrome in captive macaques. The aim of this study was to develop specific molecular diagnostic assays for M. macacae and to determine their performance characteristics. Methods We developed six real-time PCR assays on the Roche LightCycler. The accuracy, precision, selectivity, and limit of detection (LOD) were determined for each assay, in addition to further validation by testing nasal swabs from macaques presenting with epistaxis at the Tulane National Primate Research Center. Results All assays exhibited 100% specificity and were highly sensitive with an LOD of 10 fg for chromosomal assays and 1 fg for the plasmid assay. Testing of nasal swabs from 10 symptomatic macaques confirmed the presence of M. macacae in these animals. Conclusions We developed several accurate, sensitive, and species-specific real-time PCR assays for the detection of M. macacae in captive macaques. PMID:26365904
Braun, Patrick; Drago, Monica; Fanti, Diana; Fleury, Hervé; Hofmann, Jörg; Izopet, Jacques; Kühn, Sebastian; Lombardi, Alessandra; Micheli, Valeria; Sauné, Karine; Trimoulet, Pascale; Whittaker, Duncan; Artus, Alain; Rhodes, Daniel
Beckman Coulter has developed the VERIS HCV Assay for use on the new fully automated DxN VERIS Molecular Diagnostic System(¥) for HCV viral load monitoring. Evaluate the clinical performance of the new quantitative VERIS HCV Assay. Comparison was performed on 279 plasma specimens from HCV infected patients tested with the VERIS HCV Assay and COBAS(®) Ampliprep/COBAS(®) Taqman(®) HCV Test and 369 specimens tested with the VERIS HCV Assay and RealTime HCV Assay. Patient monitoring sample results from four time points were also compared. The average bias between the VERIS HCV Assay and the COBAS(®) Ampliprep/COBAS(®) Taqman(®) HCV Test was 0.04 log10IU/mL, while between the VERIS HCV Assay and the RealTime HCV Assay average bias was 0.21 log10IU/mL. Bias, however, was not consistent across the measuring range. Analysis at the lower end of quantification levels 50, 100, and 1000IU/mL showed a predicted bias for VERIS HCV Assay versus COBAS(®) Ampliprep/COBAS(®) Taqman(®) HCV Test between -0.42 and -0.22 log10IU/mL and for VERIS HCV Assay versus RealTime HCV Assay between 0.00 and 0.13 log10IU/mL. Patient monitoring of HCV viral load over time demonstrated similar levels between VERIS HCV Assay results and COBAS(®) Ampliprep/COBAS(®) Taqman(®) HCV Test (52 samples from 13 patients) and RealTime HCV Assay (112 samples from 28 patients). VERIS HCV Assay for use on the DxN VERIS Molecular Diagnostic System represents a reliable new tool for easy sample to result HCV RNA viral load monitoring. Copyright © 2017 Elsevier B.V. All rights reserved.
Citrus huanglongbing (HLB), or citrus greening, is a serious and industry-limiting disease. Preliminary diagnoses can be made through visual symptoms, and greater certainty can be achieved through quantitative real-time PCR (qPCR). Several qPCR procedures are available including those by designed by...
Gadsby, Naomi J; Hardie, Alison; Claas, Eric C J; Templeton, Kate E
The Luminex xTAG Respiratory Virus Panel (RVP) assay has been shown to offer improved diagnostic sensitivity over traditional viral culture methods and to have a sensitivity comparable to those of individual real-time nucleic acid tests for respiratory viruses. The objective of this retrospective study was to test a new, streamlined version of this assay, the RVP Fast assay, which requires considerably less run time and operator involvement. The study compared the performance of the RVP Fast assay with those of viral culture, a direct fluorescent assay (DFA), and a panel of single and multiplex real-time PCRs in the testing of 286 respiratory specimens submitted to the Edinburgh Specialist Virology Centre for routine diagnosis of viral infection between December 2007 and February 2009. At least one respiratory viral infection was detected in 13.6% of specimens by culture and DFA combined, in 49.7% by real-time PCR, and in 46.2% by the RVP Fast assay. The sensitivity and specificity of the RVP Fast assay compared to the results of real-time PCR as the gold standard were 78.8% and 99.6%, respectively. Real-time PCR-positive specimens missed by the RVP Fast assay generally had low viral loads or were positive for adenovirus. Additionally, a small number of specimens were positive by the RVP Fast assay but were not detected by real-time PCR. For some viral targets, only a small number of positive results were found in our sample set using either method; therefore, the sensitivity of detection of the RVP Fast assay for individual targets could be investigated further with a greater number of virus-positive specimens.
Kulow, Megan; Yamazaki, Wataru; Döpfer, Dörte
Bovine digital dermatitis (DD) is a severe infectious cause of lameness in cattle worldwide, with important economic and welfare consequences. There are three treponeme phylogroups (T. pedis, T. phagedenis, and T. medium) that are implicated in playing an important causative role in DD. This study was conducted to develop real-time PCR and loop-mediated isothermal amplification (LAMP) assays for the detection and differentiation of the three treponeme phylogroups associated with DD. The real-time PCR treponeme phylogroup assays targeted the 16S-23S rDNA intergenic space (ITS) for T. pedis and T. phagedenis, and the flagellin gene (flaB2) for T. medium. The 3 treponeme phylogroup LAMP assays targeted the flagellin gene (flaB2) and the 16S rRNA was targeted for the Treponeme ssp. LAMP assay. The real-time PCR and LAMP assays correctly detected the target sequence of all control strains examined, and no cross-reactions were observed, representing 100% specificity. The limit of detection for each of the three treponeme phylogroup real-time PCR and LAMP assays was ≤ 70 fg/μl. The detection limit for the Treponema spp. LAMP assay ranged from 7–690 fg/μl depending on phylogroup. Treponemes were isolated from 40 DD lesion biopsies using an immunomagnetic separation culture method. The treponeme isolation samples were then subjected to the real-time PCR and LAMP assays for analysis. The treponeme phylogroup real-time PCR and LAMP assay results had 100% agreement, matching on all isolation samples. These results indicate that the developed assays are a sensitive and specific test for the detection and differentiation of the three main treponeme phylogroups implicated in DD. PMID:28542573
Gadsby, Naomi J.; Hardie, Alison; Claas, Eric C. J.; Templeton, Kate E.
The Luminex xTAG Respiratory Virus Panel (RVP) assay has been shown to offer improved diagnostic sensitivity over traditional viral culture methods and to have a sensitivity comparable to those of individual real-time nucleic acid tests for respiratory viruses. The objective of this retrospective study was to test a new, streamlined version of this assay, the RVP Fast assay, which requires considerably less run time and operator involvement. The study compared the performance of the RVP Fast assay with those of viral culture, a direct fluorescent assay (DFA), and a panel of single and multiplex real-time PCRs in the testing of 286 respiratory specimens submitted to the Edinburgh Specialist Virology Centre for routine diagnosis of viral infection between December 2007 and February 2009. At least one respiratory viral infection was detected in 13.6% of specimens by culture and DFA combined, in 49.7% by real-time PCR, and in 46.2% by the RVP Fast assay. The sensitivity and specificity of the RVP Fast assay compared to the results of real-time PCR as the gold standard were 78.8% and 99.6%, respectively. Real-time PCR-positive specimens missed by the RVP Fast assay generally had low viral loads or were positive for adenovirus. Additionally, a small number of specimens were positive by the RVP Fast assay but were not detected by real-time PCR. For some viral targets, only a small number of positive results were found in our sample set using either method; therefore, the sensitivity of detection of the RVP Fast assay for individual targets could be investigated further with a greater number of virus-positive specimens. PMID:20357215
Nikolić, Petra; Mehle, Natasa; Gruden, Kristina; Ravnikar, Maja; Dermastia, Marina
We report here on the development of combination of assays for fast, reliable, specific and sensitive detection and discrimination of 'Candidatus Phytoplasma mali', 'Ca. P. prunorum' and 'Ca. P. pyri' from the 16Sr-X (apple proliferation - AP) group. These phytoplasmas are causal agents of diseases of fruit trees within the family Rosaceae, namely apple proliferation (AP), European stone fruit yellows (ESFY) and pear decline (PD). The designed panel of assays uses TaqMan minor groove binder probes (MGB). It comprises the same set of primers and specific probes for species-specific amplification within the 16S-23S rRNA intergenic spacer region, a set of primers and probes for amplification of the 16S ribosomal DNA region for the universal phytoplasma detection, and an additional set of primers and probe for 18S rRNA as an endogenous quality control of DNA extraction. The performance characteristics of the panel were evaluated. The advantages of new assays were shown in a comparative study with the conventional PCR, which proved their higher sensitivity combined with three-fold shorter time of testing process; and in comparison with two reported multiplex real-time PCR assays for detection of 'Ca. P. mali' or 'Ca. P. pyri'. New panel of assays were tested on the DNA samples of 'Ca. P. mali', 'Ca. P. prunorum', 'Ca. P. pyri', other phytoplasmas and other bacteria isolated from plant material. Additionally, 198 symptomatic and asymptomatic fruit tree field samples collecting during several growing seasons were tested with new assays as well. The results of this study indicate that the combination of three specific assays may be applied in routine phytoplasma surveys and in the certification programs.
Eberling, August J; Bieker-Stefanelli, Jill; Reising, Monica M; Siev, David; Martin, Barbara M; McIntosh, Michael T; Beckham, Tammy R
Classical swine fever (CSF) is an economically devastating disease of pigs. Instrumental to the control of CSF is a well-characterized assay that can deliver a rapid, accurate diagnosis prior to the onset of clinical signs. A real-time fluorogenic-probe hydrolysis (TaqMan) reverse transcription polymerase chain reaction (RT-PCR) for CSF was developed by the United States Department of Agriculture (USDA) at the Plum Island Animal Disease Center (CSF PIADC assay) and evaluated for analytical and diagnostic sensitivity and specificity. A well-characterized panel including Classical swine fever virus (CSFV), Bovine viral diarrhea virus (BVDV), and Border disease virus (BDV) isolates was utilized in initial feasibility and optimization studies. The assay was initially designed and validated for use on the ABI 7900HT using the Qiagen QuantiTect® Probe RT-PCR chemistry. However, demonstrating equivalency with multiple one-step RT-PCR chemistries and PCR platforms increased the versatility of the assay. Limit of detection experiments indicated that the Qiagen QuantiTect® Multiplex (NoROX) and the Invitrogen SuperScript® III RT-PCR kits were consistently the most sensitive one-step chemistries for use with the CSF PIADC primer/probe set. Analytical sensitivity of the CSF PIADC assay ranged from <1-2.95 log(10) TCID(50)/ml on both the ABI 7900HT and ABI 7500 platforms. The CSF PIADC assay had 100% diagnostic sensitivity and specificity when tested on a panel of 152 clinical samples from the Dominican Republic and Colombia. The ability to perform this newly developed assay in 96-well formats provides an increased level of versatility for use in CSF surveillance programs.
Takahashi, Teruyuki; Tamura, Masato; Takasu, Toshiaki
Myelitis is one of the rarest neurological complications of varicella zoster virus (VZV) infection. In this study, the authors remodeled the "wide-range" quantitative nested real-time (QNRT) polymerase chain reaction (PCR) assay to quantitatively detect a small amount of VZV-DNA in cerebrospinal fluid (CSF). For use as a specific internal control "calibrator," an original mutation-VZV (MZ) plasmid was developed. The initial copy number of VZV-DNA in CSF specimens was measured by the amplification rate of the MZ-plasmid. For 17 consecutive CSF specimens collected from three elderly patients with VZV myelitis, the diagnostic value of the wide-range QNRT-PCR assay was evaluated and compared with other conventional PCR assays and enzyme immunoassay (EIA). The MZ-plasmid demonstrated statistically uniform amplifications (F=1.016) against a wide range (1-100,000) of copy numbers of mimic VZV-DNA. The wide-range QNRT-PCR assay quantitatively and rapidly (within 48 hr) detected 5,863, 3,052, 958, and 6,721 copies/ml of VZV-DNA in the CSF specimens collected from all patients in the acute phase. Additionally, there was a significant difference (*P=0.023) in the copy number of VZV-DNA between before and after acyclovir treatment. Other conventional single PCR assays all revealed negative results, but were nevertheless time-consuming (7 days). The IgG EIA-value for VZV was continually elevated throughout the clinical course of all patients. The MZ-plasmid was thus regarded as an appropriate "calibrator" in the wide-range QNRT-PCR assay. This assay is a novel, rapid, accurate, quantitative, and highly sensitive technique, and will contribute as a reliable and useful clinical examination for the rapid diagnosis of VZV infection to central nervous system. © 2013 Wiley Periodicals, Inc.
1 Real - time PCR (RT-PCR) Assays for Burkholderia mallei and B. pseudomallei Vipin K. Rastogi1, Tu-chen Cheng1, Lisa Collins1 and Jennifer Bagley2 1...A 3. DATES COVERED - 4. TITLE AND SUBTITLE Real - time PCR (RT-PCR) Assays for Burkholderia mallei and B.pseudomallei 5a. CONTRACT NUMBER 5b...risk. There is currently no real - time PCR assay for detection of both of these pathogens. Primers and probes corresponding to specific genomic regions
Greiner, Romy; Dick, Tobias P
Hydrogen sulfide (H2S) is known to induce persulfidation of protein thiols. However, the process of H2S-induced persulfidation is not fully understood as it requires an additional oxidant. There are several mechanistic possibilities and it is of interest to determine which pathway is kinetically most relevant. Here, we detail in vitro assays for the real-time monitoring of thiol redox states in two model proteins with oxidizable cysteines, PTEN, and roGFP2. These allow kinetic measurements of the response of defined protein thiols (or disulfides) to sulfide and sulfane sulfur species. The combination of these assays with cold cyanolysis reveals the role of intermediary sulfane sulfur species in H2S-induced protein thiol oxidation.
Cecilia, D; Kakade, M; Alagarasu, K; Patil, J; Salunke, A; Parashar, D; Shah, P S
Dengue and chikungunya viruses co-circulate and cause infections that start with similar symptoms but progress to radically different outcomes. Therefore, an early diagnostic test that can differentiate between the two is needed. A single-step multiplex real-time RT-PCR assay was developed that can simultaneously detect and quantitate RNA of all dengue virus (DENV) serotypes and chikungunya virus (CHIKV). The sensitivity was 100 % for DENV and 95.8 % for CHIKV, whilst the specificity was 100 % for both viruses when compared with conventional RT-PCR. The detection limit ranged from 1 to 50 plaque-forming units. The assay was successfully used for differential diagnosis of dengue and chikungunya in Pune, where the viruses co-circulate.
Jensen, Pamela C.; Purcell, Maureen K.; Morado, J. Frank; Eckert, Ginny L.
The Alaskan red king crab (Paralithodes camtschaticus) fishery was once one of the most economically important single-species fisheries in the world, but is currently depressed. This fishery would benefit from improved stock assessment capabilities. Larval crab distribution is patchy temporally and spatially, requiring extensive sampling efforts to locate and track larval dispersal. Large-scale plankton surveys are generally cost prohibitive because of the effort required for collection and the time and taxonomic expertise required to sort samples to identify plankton individually via light microscopy. Here, we report the development of primers and a dual-labeled probe for use in a DNA-based real-time polymerase chain reaction assay targeting the red king crab, mitochondrial gene cytochrome oxidase I for the detection of red king crab larvae DNA in plankton samples. The assay allows identification of plankton samples containing crab larvae DNA and provides an estimate of DNA copy number present in a sample without sorting the plankton sample visually. The assay was tested on DNA extracted from whole red king crab larvae and plankton samples seeded with whole larvae, and it detected DNA copies equivalent to 1/10,000th of a larva and 1 crab larva/5mL sieved plankton, respectively. The real-time polymerase chain reaction assay can be used to screen plankton samples for larvae in a fraction of the time required for traditional microscopial methods, which offers advantages for stock assessment methodologies for red king crab as well as a rapid and reliable method to assess abundance of red king crab larvae as needed to improve the understanding of life history and population processes, including larval population dynamics.
Evaluation of the New BD Max GC Real-Time PCR Assay, Analytically and Clinically as a Supplementary Test for the BD ProbeTec GC Qx Amplified DNA Assay, for Molecular Detection of Neisseria gonorrhoeae
Golparian, Daniel; Boräng, Stina; Sundqvist, Martin
The new BD Max GC real-time PCR assay showed high clinical and analytical sensitivity and specificity. It can be an effective and accurate supplementary test for the BD ProbeTec GC Qx amplified DNA assay, which had suboptimal specificity, and might also be used for initial detection of Neisseria gonorrhoeae. PMID:26468501
The presentation overview is (1) Single laboratory performance assessment of human- and cattle associated PCR assays and (2) A Field Study: Evaluation of two human fecal waste management practices in Ohio watershed.
Marras, Salvatore A E; Tyagi, Sanjay; Kramer, Fred Russell
A number of formats for nucleic acid hybridization have been developed to identify DNA and RNA sequences that are involved in cellular processes and that aid in the diagnosis of genetic and infectious diseases. The introduction of hybridization probes with interactive fluorophore pairs has enabled the development of homogeneous hybridization assays for the direct identification of nucleic acids. A change in the fluorescence of these probes indicates the presence of a target nucleic acid, and there is no need to separate unbound probes from hybridized probes. The advantages of homogeneous hybridization assays are their speed and simplicity. In addition, homogeneous assays can be combined with nucleic acid amplification, enabling the detection of rare target nucleic acids. These assays can be followed in real time, providing quantitative determination of target nucleic acids over a broad range of concentrations.
The presentation overview is (1) Single laboratory performance assessment of human- and cattle associated PCR assays and (2) A Field Study: Evaluation of two human fecal waste management practices in Ohio watershed.
A real-time reverse transcription polymerase chain reaction (rtRT-PCR) assay was developed for the identification of marine vesiviruses. The primers were designed to target a 176-nucleotide fragment within a highly conserved region of the San Miguel sea lion viruses (SMSVs) capsid gene. The assay de...
In April 2013 a porcine epidemic diarrhea virus (PEDV) epidemic began in the United States. As part of the response, real-time RT-PCR assays to detect PEDV were developed by several Veterinary Diagnostic Laboratories. This study evaluated RT-PCR PEDV assays that detect the N gene (gN) and S gene (gS...
Benitez, Alvaro J; Winchell, Jonas M
We developed a single-tube multiplex real-time PCR assay capable of simultaneously detecting and discriminating Legionella spp., Legionella pneumophila, and Legionella pneumophila serogroup 1 in primary specimens. Evaluation of 21 clinical specimens and 115 clinical isolates demonstrated this assay to be a rapid, high-throughput diagnostic test with 100% specificity that may aid during legionellosis outbreaks and epidemiologic investigations.
McDowall, Rebeccah; Slavic, Durda; MacInnes, Janet I.; Cai, Hugh Y.
A real-time polymerase chain reaction (PCR) assay of the outer membrane protein (OMP) P2 gene was developed and used to test 97 putative Haemophilus parasuis pure cultures and 175 clinical tissue samples. With standard culture isolation as the gold standard, the diagnostic sensitivity and specificity of the PCR assay were determined to be 83% and 80%, respectively. PMID:24688178
Wolff, Bernard J; Benitez, Alvaro J; Desai, Heta P; Morrison, Shatavia S; Diaz, Maureen H; Winchell, Jonas M
We developed a multiplex real-time PCR assay for simultaneously detecting M. pneumoniae and typing into historically-defined P1 types. Typing was achieved based on the presence of short type-specific indels identified through whole genome sequencing. This assay was 100% specific compared to existing methods and may be useful during epidemiologic investigations.
Rios-Licea, Merab M; Bosques, Francisco J; Arroliga, Alejandro C; Galindo-Galindo, Juan O; Garza-Gonzalez, Elvira
A quadruplex real-time (RT) qPCR assay for the detection and quantification in 4h of Staphylococcusaureus, Pseudomonasaeruginosa, Acinetobacterbaumannii and Stenotrophomonasmaltophilia directly from bronchoalveolar lavage specimens was developed. The specificity of the assay was 100% for all four species. 2010 Elsevier B.V. All rights reserved.
Stefańska, Ilona; Dzieciatkowski, Tomasz; Brydak, Lidia B; Romanowska, Magdalena
This study was performed to develop real-time PCR (qPCR) for detection of human seasonal and avian influenza viruses in duplex format. First duplex qPCR detects haemagglutinin (HA) gene of influenza virus A(H1N1)pdm09 and HA gene of influenza virus A(H3N2), the second reaction detects neuraminidase (NA) gene of influenza virus A(H3N2) and NA gene of influenza virus A(H1N1)pdm09 and A(H5N1), and the third reaction detects HA gene of influenza A(H5N1) and nonstructural protein gene of influenza B virus. Primers and probes were designed using multiple alignments of target gene sequences of different reference strains. Assays were optimised for identical thermocycling conditions. Their specificity was confirmed by conventional PCR and monoplex qPCR with nucleic acids isolated from different influenza viruses and other respiratory pathogens. Plasmid constructs with a fragment of specific gene were used to assess sensitivity of the assay. The limit of detection ranged from 27 to 96 cDNA copies/reaction. Clinical specimens (n = 107) have been tested using new assays, immunofluorescence and monoplex qRT-PCR. It has been shown that developed assays have been capable of rapid and accurate simultaneous detection and differentiation of influenza viruses. They are more sensitive than immunofluorescence and at least as sensitive as monoplex qRT-PCR.
van den Brand, Marre; Peters, Remco P H; Catsburg, Arnold; Rubenjan, Anna; Broeke, Ferdi J; van den Dungen, Frank A M; van Weissenbruch, Mirjam M; van Furth, A Marceline; Kõressaar, Triinu; Remm, Maido; Savelkoul, Paul H M; Bos, Martine P
The diagnosis of late onset sepsis (LOS), a severe condition with high prevalence in preterm infants, is hampered by the suboptimal sensitivity and long turnaround time of blood culture. Detection of the infecting pathogen directly in blood by PCR would provide a much more timely result. Unfortunately, PCR-based assays reported so far are labor intensive and often lack direct species identification. Therefore we developed a real-time multiplex PCR assay tailored to LOS diagnosis which is easy-to-use, is applicable on small blood volumes and provides species-specific results within 4h. Species-specific PCR assays were selected from literature or developed using bioinformatic tools for the detection of the most prevalent etiologic pathogens: Enterococcus faecalis, Staphylococcus aureus, Staphylococcus spp., Streptococcus agalactiae, Escherichia coli, Pseudomonas aeruginosa, Klebsiella spp. and Serratia marcescens. The PCR assays showed 100% specificity, full coverage of the target pathogens and a limit of detection (LOD) of ≤10CFUeq./reaction. These LOD values were maintained in the multiplex format or when bacterial DNA was isolated from blood. Clinical evaluation showed high concordance between the multiplex PCR and blood culture. In conclusion, we developed a multiplex PCR that allows the direct detection of the most important bacterial pathogens causing LOS in preterm infants.
Tseng, Hubert; Gage, Jacob A.; Shen, Tsaiwei; Haisler, William L.; Neeley, Shane K.; Shiao, Sue; Chen, Jianbo; Desai, Pujan K.; Liao, Angela; Hebel, Chris; Raphael, Robert M.; Becker, Jeanne L.; Souza, Glauco R.
An ongoing challenge in biomedical research is the search for simple, yet robust assays using 3D cell cultures for toxicity screening. This study addresses that challenge with a novel spheroid assay, wherein spheroids, formed by magnetic 3D bioprinting, contract immediately as cells rearrange and compact the spheroid in relation to viability and cytoskeletal organization. Thus, spheroid size can be used as a simple metric for toxicity. The goal of this study was to validate spheroid contraction as a cytotoxic endpoint using 3T3 fibroblasts in response to 5 toxic compounds (all-trans retinoic acid, dexamethasone, doxorubicin, 5′-fluorouracil, forskolin), sodium dodecyl sulfate (+control), and penicillin-G (−control). Real-time imaging was performed with a mobile device to increase throughput and efficiency. All compounds but penicillin-G significantly slowed contraction in a dose-dependent manner (Z’ = 0.88). Cells in 3D were more resistant to toxicity than cells in 2D, whose toxicity was measured by the MTT assay. Fluorescent staining and gene expression profiling of spheroids confirmed these findings. The results of this study validate spheroid contraction within this assay as an easy, biologically relevant endpoint for high-throughput compound screening in representative 3D environments. PMID:26365200
Gatto, Francesca; Cassina, Giulia; Broccolo, Francesco; Morreale, Giuseppe; Lanino, Edoardo; Di Marco, Eddi; Vardas, Efthiya; Bernasconi, Daniela; Buttò, Stefano; Principi, Nicola; Esposito, Susanna; Scarlatti, Gabriella; Lusso, Paolo; Malnati, Mauro S
Accurate and highly sensitive tests for the diagnosis of active Epstein-Barr virus (EBV) infection are essential for the clinical management of individuals infected with EBV. A calibrated quantitative real-time PCR assay for the measurement of EBV DNA of both EBV-1 and 2 subtypes was developed, combining the detection of the EBV DNA and a synthetic DNA calibrator in a multiplex PCR format. The assay displays a wide dynamic range and a high degree of accuracy even in the presence of 1μg of human genomic DNA. This assay measures with the same efficiency EBV DNA from strains prevalent in different geographic areas. The clinical sensitivity and specificity of the system were evaluated by testing 181 peripheral blood mononuclear cell (PBMCs) and plasma specimens obtained from 21 patients subjected to bone marrow transplantation, 70 HIV-seropositive subjects and 23 healthy controls. Patients affected by EBV-associated post-transplant lymphoprolipherative disorders had the highest frequency of EBV detection and the highest viral load. Persons infected with HIV had higher levels of EBV DNA load in PBMCs and a higher frequency of EBV plasma viremia compared to healthy controls. In conclusion, this new assay provides a reliable high-throughput method for the quantitation of EBV DNA in clinical samples.
Liu, Qingtian; Yang, Zengqi; Hao, Huafang; Cheng, Shenli; Fan, Wentao; Du, Enqi; Xiao, Sa; Wang, Xinglong; Zhang, Shuxia
Avian encephalomyelitis virus (AEV) causes epidemic diseases in poultry worldwide. A SYBR Green real-time reverse transcription-polymerase chain reaction (rRT-PCR) assay was developed for the rapid detection and quantitation of AEV in this study. A pair of specific primers was designed in the highly conserved VP1 gene of this virus. When comparing this assay with conventional RT-PCR, the rRT-PCR assay was 100 times more sensitive and could detect levels as low as 10 standard DNA copies of the AEV SX strain. The specificity of this technique was evaluated in five other avian pathogens. The AEV RNA was detected as early as three days post-infection in chicken embryos. All 18 clinical chicken brains collected from an AEV outbreak in Northwestern China were detected to be positive (100%) using the rRT-PCR assay. However, only 5 of the 18 samples were positive (28%) using the conventional RT-PCR. The results were confirmed by virus isolation in chicken embryos. This high sensitivity, specificity, and simplicity of the SYBR Green rRT-PCR approach can be a more effective method than the conventional one for AEV diagnosis and surveillance.
Wang, Fei; Tian, Yin; Yang, Jing; Sun, Fu-Jun; Sun, Ning; Liu, Bi-Yong; Tian, Rui; Ge, Guang-Lu; Zou, Ming-qiang; Deng, Cong-liang; Liu, Yi
To establish a magnetic nanoparticles separation-based quantitative real-time PCR (RT-PCR) assay for fast and accurate detection of Plasmodium falciparum and providing a technical support for improving the control and prevention of imported malaria. According to the conserved sequences of the P. falciparum genome 18SrRNA, the species-specific primers and probe were designed and synthetized. The RT-PCR was established by constructing the plasmid standard, fitting the standard curve and using magnetic nanoparticles separation. The sensitivity and specificity of the assay were evaluated. The relationship between the threshold cycle (Ct) and logarithm of initial templates copies was linear over a range of 2.5 x 10(1) to 2.5 x 10(8) copies/μl (R2 = 0.999). Among 13 subjects of entry frontier, a P. falciparum carrier with low load was detected by using the assay and none was detected with the conventional examinations (microscopic examinations and rapid tests). This assay shows a high sensitivity in detection of P. falciparum, with rapid and accurate characteristics, and is especially useful in diagnosis of P. falciparum infectors with low parasitaemia at entry-exit frontier ports.
Kuo, Hsiao-Che; Wang, Ting-Yu; Chen, Peng-Peng; Chen, Young-Mao; Chuang, Hui-Ching; Chen, Tzong-Yueh
Viral nervous necrosis caused by nervous necrosis virus (NNV) exacts a high mortality and results in huge economic losses in grouper aquaculture in Taiwan. The present study developed a real-time quantitative PCR (qPCR) method for NNV monitoring. The assay showed a strong linear correlation (r2 = 0.99) between threshold cycle (CT) and RNA quantities, which allowed identification of infected groupers by the CT value and could be exploited to warn of NNV infection prior to an outbreak in grouper fish farms. Real-time qPCR also confirmed the copious content of NNV in grouper fin, similar to that in primary tissues; the result was verified by using in situ reverse transcription-PCR (RT-PCR). This indicated that grouper fin was a suitable sample for NNV detection, in a manner that could be relatively benign to the fish. The rapid spread of NNV infection to the entire population of affected farms was evident. The developed real-time qPCR method is rapid, highly sensitive, and applicable to routine high-throughput detection of large numbers of samples and has potential as a suitable tool for diagnostic, epidemiological, and genetic studies of grouper aquaculture. PMID:21233077
Mieog, Jos C; Howitt, Crispin A; Ral, Jean-Philippe
A crucial step in the evaluation of newly produced transgenic plants is the selection of homozygous plants. Here we describe an efficient and highly flexible real-time PCR-based method for the development of homozygous lines in plant models with complex (multiple) genomes and/or relatively long generation times (>3 months) using direct copy number determinations. An existing DNA extraction method was converted into a high-throughput plant leaf DNA extraction procedure yielding DNA suitable for real-time PCR analyses. Highly specific and efficient primer pairs were developed for a bread wheat reference gene (Epsilon Cyclase) and for standard sequence elements in the gene cassette routinely used for cereal transformations (an intron bridge and the Nopaline Synthase terminator). The real-time PCR assay reliably distinguished wheat plants with a single copy of the transgene from individuals with multiple copies or those lacking the transgene. To obtain homozygous lines carrying a unique insertion event as efficiently as possible, T0 plants (plants raised from transformed callus) with a single copy of the transgene were selected and their progeny screened for homozygous plants. Finally, the assay was adapted to work on rice. The ability to quickly, easily and accurately quantify the construct copy numbers, as provided by the real-time PCR assay, greatly improved the efficiency and reliability of the selection of homozygous transgenic plants in our case study. We were able to select homozygous plants in early generations, avoiding time-consuming methods such as large scale analysis of segregation patterns of descendants and/or Southern blotting. Additionally, the ability to specifically develop homozygous lines carrying a unique insertion event could be important in avoiding gene silencing due to co-suppression, and if needed assist in the selection of lines suitable for future deregulation. The same primer pairs can be used to quantify many different wheat transgenic
Dolz, Sandra; Barragán, Eva; Fuster, Óscar; Llop, Marta; Cervera, José; Such, Esperanza; De Juan, Inmaculada; Palanca, Sarai; Murria, Rosa; Bolufer, Pascual; Luna, Irene; Gómez, Inés; López, María; Ibáñez, Mariam; Sanz, Miguel A
The recent World Health Organization classification recognizes different subtypes of acute myeloid leukemia (AML) according to the presence of several recurrent genetic abnormalities. Detection of these abnormalities and other molecular changes is of increasing interest because it contributes to a refined diagnosis and prognostic assessment in AML and enables monitoring of minimal residual disease. These genetic abnormalities can be detected using single RT-PCR, although the screening is still labor intensive and costly. We have developed a novel real-time RT-PCR assay to simultaneously detect 15 AML-associated rearrangements that is a simple and easily applicable method for use in clinical diagnostic laboratories. This method showed 100% specificity and sensitivity (95% confidence interval, 91% to 100% and 92% to 100%, respectively). The procedure was validated in a series of 105 patients with AML. The method confirmed all translocations detected using standard cytogenetics and fluorescence in situ hybridization and some additional undetected rearrangements. Two patients demonstrated two molecular rearrangements simultaneously, with BCR-ABL1 implicated in both, in addition to RUNX1-MECOM in one patient and PML-RARA in another. In conclusion, this novel real-time RT-PCR assay for simultaneous detection of multiple AML-associated fusion genes is a versatile and sensitive method for reliable screening of recurrent rearrangements in AML.
Background Tuber magnatum, the Italian white truffle, is the most sought-after edible ectomycorrhizal mushroom. Previous studies report the difficulties of detecting its mycorrhizas and the widespread presence of its mycelium in natural production areas, suggesting that the soil mycelium could be a good indicator to evaluate its presence in the soil. In this study a specific real-time PCR assay using TaqMan chemistry was developed to detect and quantify T. magnatum in soil. This technique was then applied to four natural T. magnatum truffières located in different regions of Italy to validate the method under different environmental conditions. Results The primer/probe sets for the detection and quantification of T. magnatum were selected from the ITS rDNA regions. Their specificity was tested in silico and using qualitative PCR on DNA extracted from 25 different fungal species. The T. magnatum DNA concentration was different in the four experimental truffières and higher in the productive plots. T. magnatum mycelium was however also detected in most of the non-productive plots. Ascoma production during the three years of the study was correlated with the concentration of T. magnatum DNA. Conclusions Taken together, these results suggest that the specific real-time PCR assay perfected in this study could be an useful tool to evaluate the presence and dynamics of this precious truffle in natural and cultivated truffières. PMID:22672347
Downing, Nicholas S; Ross, Joseph S; Jackevicius, Cynthia A; Krumholz, Harlan M
The ongoing debate concerning the efficacy of fenofibrate has overshadowed an important aspect of the drug's history: Abbott Laboratories, the maker of branded fenofibrate, has produced several bioequivalent reformulations that dominate the market, although generic fenofibrate has been available for almost a decade. This continued use of branded formulations, which cost twice as much as generic versions of fenofibrate, imposes an annual cost of approximately $700 million on the US health care system. Abbott Laboratories maintained its dominance of the fenofibrate market in part through a complex switching strategy involving the sequential launch of branded reformulations that had not been shown to be superior to the first-generation product and patent litigation that delayed the approval of generic formulations. The small differences in dose of the newer branded formulations prevented their substitution with generics of older-generation products. As soon as direct generic competition seemed likely at the new dose level, where substitution would be allowed, Abbott would launch another reformulation, and the cycle would repeat. Based on the fenofibrate example, our objective is to describe how current policy can allow pharmaceutical companies to maintain market share using reformulations of branded medications, without demonstrating the superiority of next-generation products.
Smith, Darci R; Lee, John S; Jahrling, Jordan; Kulesh, David A; Turell, Michael J; Groebner, Jennifer L; O'Guinn, Monica L
Chikungunya (CHIK) and O'nyong-nyong (ONN) are important emerging arthropod-borne diseases. Molecular diagnosis of these two viruses in mosquitoes has not been evaluated, and the effects of extraneous mosquito tissue on assay performance have not been tested. Additionally, no real-time reverse transcription-polymerase chain reaction (RT-PCR) assay exists for detecting ONN virus (ONNV) RNA. We describe the development of sensitive and specific real-time RT-PCR assays for detecting CHIK and ONN viral RNA in mosquitoes, which have application for field use. In addition, we compared three methods for primer/probe design for assay development by evaluating their sensitivity and specificity. This comparison resulted in development of virus-specific assays that could detect less than one plaque-forming unit equivalent of each of the viruses in mosquitoes. The use of these assays will aid in arthropod-borne disease surveillance and in the control of the associated diseases.
Liew, P S; Teh, C S J; Lau, Y L; Thong, K L
Shigellosis is a foodborne illness caused by the genus Shigella and is an important global health issue. The development of effective techniques for rapid detection of this pathogen is essential for breaking the chain of transmission. Therefore, we have developed a novel loop-mediated isothermal amplification (LAMP) assay targeting the invasion plasmid antigen H (ipaH) gene to rapidly detect Shigella species. This assay could be performed in 90 min at an optimal temperature of 64ºC, with endpoint results visualized directly. Notably, the method was found to be more sensitive than conventional PCR. Indeed, the detection limit for the LAMP assay on pure bacterial cultures was 5.9 x 10(5) CFU/ml, while PCR displayed a limit of 5.9 x 10(7) CFU/ml. In spiked lettuce samples, the sensitivity of the LAMP assay was 3.6 x 10(4) CFU/g, whereas PCR was 3.6 x 10(5) CFU/g. Overall, the assay accurately identified 32 Shigella spp. with one enteroinvasive Escherichia coli displaying positive reaction while the remaining 32 non-Shigella strains tested were negative.
Background Gene expression analysis is an important tool in contemporary research, with real-time PCR as the method of choice for quantifying transcription levels. Co-analysis of suitable reference genes is crucial for accurate expression normalisation. Reference gene expression may vary, e.g., among species or tissues; thus, candidate genes must be tested prior to use in expression studies. The domestic cat is an important study subject in both medical research and veterinary medicine. The aim of the present study was to develop TaqMan® real-time PCR assays for eight potential reference genes and to test their applicability for feline samples, including blood, lymphoid, endocrine, and gastrointestinal tissues from healthy cats, and neoplastic tissues from FeLV-infected cats. Results RNA extraction from tissues was optimised for minimal genomic DNA (gDNA) contamination without use of a DNase treatment. Real-time PCR assays were established and optimised for v-abl Abelson murine leukaemia viral oncogene homolog (ABL), β-actin (ACTB), β-2-microglobulin (B2M), β-glucuronidase (GUSB), hydroxymethyl-bilane synthase (HMBS), hypoxanthine phosphoribosyltransferase (HPRT), ribosomal protein S7 (RPS7), and tryptophan 5-monooxygenase activation protein, zeta polypeptide (YWHAZ). The presence of pseudogenes was confirmed for four of the eight investigated genes (ACTB, HPRT, RPS7, and YWHAZ). The assays were tested together with previously developed TaqMan® assays for feline glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the universal 18S rRNA gene. Significant differences were found among the expression levels of the ten candidate reference genes, with a ~106-fold expression difference between the most abundant (18S rRNA) and the least abundant genes (ABL, GUSB, and HMBS). The expression stability determined by the geNorm and NormFinder programs differed significantly. Using the ANOVA-based NormFinder program, RPS7 was the most stable gene in the tissues studied
Nam, Hyang-Mi; Srinivasan, Velusamy; Gillespie, Barbara E; Murinda, Shelton E; Oliver, Stephen P
The objective of this study was to develop and evaluate a SYBR Green 1 real-time PCR method for the specific detection of Salmonella spp. in dairy farm environmental samples. Previously reported 119-bp invA gene was selected for specificity, and 124 Salmonella spp. including type strains and 116 non-Salmonella strains were evaluated. All Salmonella strains tested were invA-positive and all non-salmonella strains yielded no amplification products. The melting temperature (Tm=79 degrees C) was consistently specific for the amplicon. Correlation coefficients of standard curves constructed using the threshold cycle (C(T)) versus copy numbers of Salmonella Enteritidis showed good linearity in broth (R2=0.994; slope=3.256) and sterilized milk (R2=0.988; slope=3.247), and the minimum levels of detection were >10(2) and >10(3) colony forming units (CFU)/ml, respectively. To validate the real-time PCR assay, an experiment was conducted with both spiked and naturally contaminated samples. Lagoon water, feed/silage, bedding soil, and bulk tank milk samples obtained from dairy farms were spiked with 10(0) to 10(5) CFU/ml of Salmonella Enteritidis. Sensitivities for detecting Salmonella in these sources were 10(3) to 10(4) CFU/ml of inoculums in broth without enrichment. Detection limits were reduced to <10 CFU/ml of inoculum in broth after 18 h enrichment. Ninety-three environmental samples including fecal slurry, feed/silage, lagoon water, drinking water, bulk tank milk, farm soil, and bedding soil were analyzed for the presence of Salmonella by real-time PCR, results were compared with those obtained by conventional culture methods. All samples analyzed were negative for Salmonella by both real-time PCR and standard culture method. No false positive or false negative results were detected.
Cheng, Jiaoying; Bian, Meilu; Cong, Xiao; Sun, Aiping; Li, Min; Ma, Li; Chen, Ying; Liu, Jun
It has been confirmed that detection of high-risk human papillomavirus (HR HPV) DNA is useful in cervical cancer (CC) screening. Recently, a new real-time fluorescent polymerase chain reaction (PCR) assay was developed to detect HR HPV. This assay can synchronize nucleic acid amplification and testing using specific primers for 13 types of HR HPV genomes, combined with specific TaqMan fluorescent marker probe techniques through the fluorescence automatic PCR instrument. Furthermore, it uses TaqGold™ DNA polymerase, which minimizes the amount of non-specific amplification and increases the sensitivity of the assay. The aim of this study was to evaluate the analytical and clinical performance of the real-time fluorescent PCR assay in CC screening, compared to the Qiagen Hybrid Capture(®) II High-Risk HPV DNA test(®) (HC II). In total, 1,252 cervical specimens were collected from women between 19 and 71 years of age. The specimens were examined with three different assays, real-time fluorescent PCR assay and HC II for HR HPV detection combined with liquid-based cytology. Women with cytological abnormalities or HR HPV-positive results underwent colposcopy and cervical biopsy. This study demonstrated good overall agreement between HC II and real-time fluorescent PCR assay (overall agreement, 92.25%; Cohen's κ=0.814). For the detection of high-grade cervical intraepithelial neoplasias (CIN) and CC, the sensitivity of HC II and real-time fluorescent PCR was 94.48 and 92.82%, respectively, and the negative predictive value was 98.85 and 98.54%, respectively. High HR HPV infection rate of the high-grade CIN and CC group was detected (P<0.05). In conclusion, real-time fluorescent PCR assay provides similar results compared to the HC II test for HR HPV detection and could be used in CC screening in clinic.
CHENG, JIAOYING; BIAN, MEILU; CONG, XIAO; SUN, AIPING; LI, MIN; MA, LI; CHEN, YING; LIU, JUN
It has been confirmed that detection of high-risk human papillomavirus (HR HPV) DNA is useful in cervical cancer (CC) screening. Recently, a new real-time fluorescent polymerase chain reaction (PCR) assay was developed to detect HR HPV. This assay can synchronize nucleic acid amplification and testing using specific primers for 13 types of HR HPV genomes, combined with specific TaqMan fluorescent marker probe techniques through the fluorescence automatic PCR instrument. Furthermore, it uses TaqGold™ DNA polymerase, which minimizes the amount of non-specific amplification and increases the sensitivity of the assay. The aim of this study was to evaluate the analytical and clinical performance of the real-time fluorescent PCR assay in CC screening, compared to the Qiagen Hybrid Capture® II High-Risk HPV DNA test® (HC II). In total, 1,252 cervical specimens were collected from women between 19 and 71 years of age. The specimens were examined with three different assays, real-time fluorescent PCR assay and HC II for HR HPV detection combined with liquid-based cytology. Women with cytological abnormalities or HR HPV-positive results underwent colposcopy and cervical biopsy. This study demonstrated good overall agreement between HC II and real-time fluorescent PCR assay (overall agreement, 92.25%; Cohen’s κ=0.814). For the detection of high-grade cervical intraepithelial neoplasias (CIN) and CC, the sensitivity of HC II and real-time fluorescent PCR was 94.48 and 92.82%, respectively, and the negative predictive value was 98.85 and 98.54%, respectively. High HR HPV infection rate of the high-grade CIN and CC group was detected (P<0.05). In conclusion, real-time fluorescent PCR assay provides similar results compared to the HC II test for HR HPV detection and could be used in CC screening in clinic. PMID:24648936
Swan, Heather; Sloan, Lynne; Muyombwe, Anthony; Chavalitshewinkoon-Petmitr, Porntip; Krudsood, Srivicha; Leowattana, Wattana; Wilairatana, Polrat; Looareesuwan, Sornchai; Rosenblatt, Jon
We compared the diagnosis of malaria in 297 patients from Thailand by a real-time polymerase chain reaction (PCR) assay using the LightCycler with conventional microscopy using Giemsa-stained thick and thin blood films. The PCR assay can be completed in one hour and has the potential to detect and identify four species of Plasmodium in a single reaction by use of melting temperature curve analysis (however, we did not detect Plasmodium ovale in this study). Blood was collected, stored, and transported on IsoCode STIX, which provide a stable matrix for the archiving and rapid simple extraction of DNA. A genus-specific primer set corresponding to the 18S ribosomal RNA was used to amplify the target sequence. Fluorescence resonance energy technology hybridization probes were designed for P. falciparum over a region containing basepair mismatches, which allowed differentiation of the other Plasmodium species. The PCR results correlated with the microscopic results in 282 (95%) of 297 patient specimens. Most of these were single-species infections caused by P. vivax (150) and P. falciparum (120), along with 5 P. malariae, 2 mixed infections (P. falciparum and P. vivax), and 5 negative specimens. No negative microscopy specimens were positive by PCR (100% specificity for detection of any Plasmodium). The 15 discrepant results could not be resolved, but given the subjective nature of microscopy and the analytical objectivity of the PCR, the PCR results may be correct. The ability of the PCR method to detect mixed infections or to detect P. ovale could not be determined in this study. Within the limitations of initial equipment costs, this real-time PCR assay is a rapid, accurate, and efficient method for the specific diagnosis of malaria. It may have application in clinical laboratories, as well as in epidemiologic studies and antimalarial efficacy trials.
Zeschnigk, Michael; Böhringer, Stefan; Price, Elizabeth Ann; Onadim, Zerrin; Masshöfer, Lars; Lohmann, Dietmar R
Altered methylation patterns have been found to play a role in developmental disorders, cancer and aging. Increasingly, changes in DNA methylation are used as molecular markers of disease. Therefore, there is a need for reliable and easy to use techniques to detect and measure DNA methylation in research and routine diagnostics. We have established a novel quantitative analysis of methylated alleles (QAMA) which is essentially a major improvement over a previous method based on real-time PCR (MethyLight). This method is based on real-time PCR on bisulfite-treated DNA. A significant advantage over conventional MethyLight is gained by the use of TaqMan probes based on minor groove binder (MGB) technology. Their improved sequence specificity facilitates relative quantification of methylated and unmethylated alleles that are simultaneously amplified in single tube. This improvement allows precise measurement of the ratio of methylated versus unmethylated alleles and cuts down potential sources of inter-assay variation. Therefore, fewer control assays are required. We have used this novel technical approach to identify hypermethylation of the CpG island located in the promoter region of the retinoblastoma (RB1) gene and found that QAMA facilitates reliable and fast measurement of the relative quantity of methylated alleles and improves handling of diagnostic methylation analysis. Moreover, the simplified reaction setup and robustness inherent to the single tube assay facilitates high-throughput methylation analysis. Because the high sequence specificity inherent to the MGB technology is widely used to discriminate single nucleotide polymorphisms, QAMA potentially can be used to discriminate the methylation status of single CpG dinucleotides.
Geets, Joke; de Cooman, Michaël; Wittebolle, Lieven; Heylen, Kim; Vanparys, Bram; De Vos, Paul; Verstraete, Willy; Boon, Nico
In order to improve wastewater treatment processes, a need exists for tools that rapidly give detailed insight into the community structure of activated sludge, supplementary to chemical and physical data. In this study, the advantages of microarrays and quantitative polymerase chin reaction (PCR) methods were combined into a real-time PCR assay that allows the simultaneous quantification of phylogenetic and functional genes involved in nitrification and denitrification processes. Simultaneous quantification was possible along a 5-log dynamic range and with high linear correlation (R (2) > 0.98). The specificity of the assay was confirmed by cloning and sequencing analyses of PCR amplicons obtained from activated sludge. The real-time assay was validated on mixed liquid samples of different treatment plants, which varied in nitrogen removal rate. The abundance of ammonia oxidizers was in the order of magnitude of 10(6) down to 10(4) ml(-1), whereas nitrite oxidizers were less abundant (10(3)-10(1) order of magnitude). The results were in correspondence with the nitrite oxidation rate in the sludge types. As for the nirS, nirK, and nosZ gene copy numbers, their abundance was generally in the order of magnitude of 10(8)-10(5). When sludge samples were subjected to lab-scale perturbations, a decrease in nitrification rate was reflected within 18 h in the copy numbers of nitrifier genes (decrease with 1 to 5 log units), whereas denitrification genes remained rather unaffected. These results demonstrate that the method is a fast and accurate tool for the analysis of the (de)nitrifying community structure and size in both natural and engineered environmental samples.
Yu, Haijie; Huang, Bin; Zhuo, Xunhui; Chen, Xueqiu; Du, Aifang
Real-time PCR-based detection of Toxoplasma gondii is very sensitive and convenient for diagnosing toxoplasmosis. However, the performance of the PCR assays could be influenced by the target gene chosen. Here we evaluate a real-time PCR assay using double-stranded DNA dyes (SYBR(®) Green I assay) with a new set of primers targeting the SAG1 gene for the fast and specific detection of T. gondii. The assay showed higher sensitivity than conventional PCR protocols using T. gondii DNA as template. The detection limit of the developed real-time PCR assay was in the order of 1 tachyzoite. The assay was also assessed by experimentally infected mice and showed positive results for blood (25%), spleen (50%) and lung (50%) as early as 1 dpi. The specificity of the assay was confirmed by using DNA from Neospora caninum, Escherichia coli, Babesia bovis, Trypanosoma brucei, Cryptosporidium parvum, and Toxocara canis. Assay applicability was successfully tested in blood samples collected from slaughtered pigs. These results indicate that, based on SYBR(®) green I, the quantitative SAG1 assay may also be useful in the study of the pathogenicity, immunoprophylaxis, and treatment of T. gondii.
Özeş, Ali R; Feoktistova, Kateryna; Avanzino, Brian C; Baldwin, Enoch P; Fraser, Christopher S
Many physiological functions of helicases are dependent on their ability to unwind nucleic acid duplexes in an ATP-dependent fashion. Determining the kinetic frameworks of these processes is crucial to understanding how these proteins function. We recently developed a fluorescence assay to monitor RNA duplex unwinding by DEAD-box helicases in real time. In this assay, two fluorescently modified short reporter oligonucleotides are annealed to an unmodified RNA loading strand of any length so that the fluorescent moieties of the two reporters find themselves in close proximity to each other and fluorescence is quenched. One reporter is modified with cyanine 3 (Cy3), whereas the other is modified with a spectrally paired black-hole quencher (BHQ). As the helicase unwinds the loading strand, the enzyme displaces the Cy3-modified reporter, which will bind to a capture or competitor DNA strand, permanently separating it from the BHQ-modified reporter. Complete separation of the Cy3-modified reporter strand is thus detected as an increase in total fluorescence. This assay is compatible with reagentless biosensors to monitor ATPase activity so that the coupling between ATP hydrolysis and duplex unwinding can be determined. With the protocol described, obtaining data and analyzing results of unwinding and ATPase assays takes ∼4 h.
Robert-Gangneux, Florence; Brenier-Pinchart, Marie-Pierre; Yera, Hélène; Belaz, Sorya; Varlet-Marie, Emmanuelle; Bastien, Patrick
Molecular diagnosis of congenital toxoplasmosis or disseminated toxoplasmosis is mainly based on PCR. The repeated DNA element rep529 has become the main DNA target used in most, whether laboratory-developed or commercial, PCR methods. In this multicenter study, we evaluated the Toxoplasma ELITe MGB® (Elitech®) commercial kit, by comparison with three reference quantitative PCR assays (RA) used in routine in three proficient laboratories of the French National Reference Center for Toxoplasmosis network, using Toxoplasma calibrated suspensions diluted to obtain a range of concentrations from 0.1 to 10,000 parasites/mL. These suspensions were extracted either with the DNA extraction kit (EXTRAblood®, Elitech®) recommended by the manufacturer or the QIAamp DNA-minikit (Qiagen). The Toxoplasma ELITe MGB® assay was also evaluated on a panel of 128 clinical samples, including 56 amniotic fluid, 55 placenta, and various other samples, of which 95 originated from patients with proven toxoplasmosis. The ELITe MGB assay amplified less frequently low-concentration replicates (<10 parasites/mL) of calibrated suspensions, than the RA of 2/3 laboratories. Additionally, the combination EXTRAblood®/Toxoplasma ELITe MGB® yielded poorer sensitivity than the combination QIAmp DNA-minikit/ELITe MGB® for low parasite concentrations (p<0.001 for 1 parasite/mL). On clinical samples, the sensitivity and the specificity of the commercial assay were 89% and 100%, respectively. The sensitivity ranged from 79% to 100% with placenta and amniotic fluid samples, respectively. Overall, this study shows that the Toxoplasma ELITe MGB® assay is suitable for the diagnosis of toxoplasmosis from non-cell-rich or non-hemoglobin-rich samples, and that the EXTRAblood® kit is not optimal.
Peng, Jingfu; Yu, Xiaoli; Cui, Zhenling; Xue, Wenfei; Luo, Ziyi; Wen, Zilu; Liu, Minghua; Jiang, Danqing; Zheng, Heping; Wu, Hai; Zhang, Shulin; Li, Yao
Background: Failure to early detect multidrug-resistant tuberculosis (MDR-TB) results in treatment failure and poor clinical outcomes, and highlights the need to rapidly detect resistance to rifampicin (RIF) and isoniazid (INH). Methods: In Multi-Fluorescence quantitative Real-Time PCR (MF-qRT-PCR) assay, 10 probes labeled with four kinds of fluorophores were designed to detect the mutations in regions of rpoB, katG, mabA-inhA, oxyR-ahpC, and rrs. The efficiency of MF-qRT-PCR assay was tested using 261 bacterial isolates and 33 clinical sputum specimens. Among these samples, 227 Mycobacterium tuberculosis isolates were analyzed using drug susceptibility testing (DST), DNA sequencing and MF-qRT-PCR assay. Results: Compared with DST, MF-qRT-PCR sensitivity and specificity for RIF-resistance were 94.6 and 100%, respectively. And the detection sensitivity and specificity for INH-resistance were 85.9 and 95.3%, respectively. Compared with DNA sequencing, the sensitivity and specificity of our assay were 97.2 and 100% for RIF-resistance and 97.9 and 96.4% for INH-resistance. Compared with Phenotypic strain identification, MF-qRT-PCR can distinguish 227 M. tuberculosis complexes (MTC) from 34 Non-tuberculous mycobacteria (NTM) isolates with 100% accuracy rate. Conclusions: MF-qRT-PCR assay was an efficient, accurate, reliable, and easy-operated method for detection of RIF and INH-resistance, and distinction of MTC and NTM of clinical isolates. PMID:27199947
Rawsthorne, Helen; Phister, Trevor G
Zygosaccharomyces bailii is a major food and beverage spoilage organism. Existing methods for its detection involve lengthy enrichment techniques and then the result does not always differentiate between Z. bailii and Saccharomyces cerevisiae. In this work, we developed a quantitative real-time PCR assay for the rapid detection of Z. bailii from fruit juices and wine even in the presence of non-target DNA. Primers were designed to the gene coding for the D1/D2 loop of the 26S ribosomal RNA subunit producing a single PCR product with a melting temperature of 83.5 degrees C. As few as 2 cells per ml could be detected by the assay in cranberry raspberry and apple juices and 22 cells per ml from grape juice. The assay was equally efficient in wine, detecting 6 cells per ml even in the presence of 10(7)S. cerevisiae cells. The CFU/ml as determined by plating on YM media showed excellent correlation with the CFU/ml established by the QPCR assay for all the beverages examined. Unknown samples of Z. bailii were prepared in the juices and wine and examined by QPCR. The QPCR estimated cell number was in good agreement with the cell counts obtained by plating, the exception being the cranberry raspberry juice sample. It was determined by live/dead cell counts that the Z. bailii cells were less viable in this juice thus leading to an overestimation of CFU/ml by QPCR. However, the correlation was high between QPCR and total cell count as determined by fluorescent microscopy. This assay provides a rapid and accurate method to establish the levels of the total Z. bailii population which consists of both viable and nonviable cells.
Chen, Liang; Mediavilla, José R; Endimiani, Andrea; Rosenthal, Marnie E; Zhao, Yanan; Bonomo, Robert A; Kreiswirth, Barry N
Carbapenem resistance mediated by plasmid-borne Klebsiella pneumoniae carbapenemases (KPC) is an emerging problem of significant clinical importance in Gram-negative bacteria. Multiple KPC gene variants (bla(KPC)) have been reported, with KPC-2 (bla(KPC-2)) and KPC-3 (bla(KPC-3)) associated with epidemic outbreaks in New York City and various international settings. Here, we describe the development of a multiplex real-time PCR assay using molecular beacons (MB-PCR) for rapid and accurate identification of bla(KPC) variants. The assay consists of six molecular beacons and two oligonucleotide primer pairs, allowing for detection and classification of all currently described bla(KPC) variants (bla(KPC-2) to bla(KPC-11)). The MB-PCR detection limit was 5 to 40 DNA copies per reaction and 4 CFU per reaction using laboratory-prepared samples. The MB-PCR probes were highly specific for each bla(KPC) variant, and cross-reactivity was not observed using DNA isolated from several bacterial species. A total of 457 clinical Gram-negative isolates were successfully characterized by our MB-PCR assay, with bla(KPC-3) and bla(KPC-2) identified as the most common types in the New York/New Jersey metropolitan region. The MB-PCR assay described herein is rapid, sensitive, and specific and should be useful for understanding the ongoing evolution of carbapenem resistance in Gram-negative bacteria. As novel bla(KPC) variants continue to emerge, the MB-PCR assay can be modified in response to epidemiologic developments.
Lin, Chih-Hui; Chen, Yu-Chieh; Pan, Tzu-Ming
Quantitative real-time PCR (qPCR) is the gold standard for the quantification of specific nucleic acid sequences. However, a serious concern has been revealed in a recent report: supercoiled plasmid standards cause significant over-estimation in qPCR quantification. In this study, we investigated the effect of plasmid DNA conformation on the quantification of DNA and the efficiency of qPCR. Our results suggest that plasmid DNA conformation has significant impact on the accuracy of absolute quantification by qPCR. DNA standard curves shifted significantly among plasmid standards with different DNA conformations. Moreover, the choice of DNA measurement method and plasmid DNA conformation may also contribute to the measurement error of DNA standard curves. Due to the multiple effects of plasmid DNA conformation on the accuracy of qPCR, efforts should be made to assure the highest consistency of plasmid standards for qPCR. Thus, we suggest that the conformation, preparation, quantification, purification, handling, and storage of standard plasmid DNA should be described and defined in the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) to assure the reproducibility and accuracy of qPCR absolute quantification. PMID:22194997
Volle, Romain; Nourrisson, Céline; Mirand, Audrey; Regagnon, Christel; Chambon, Martine; Henquell, Cécile; Bailly, Jean-Luc; Peigue-Lafeuille, Hélène; Archimbaud, Christine
Human enteroviruses are the most frequent cause of aseptic meningitis and are involved in other neurological infections. Qualitative detection of enterovirus genomes in cerebrospinal fluid is a prerequisite in diagnosing neurological diseases. The pathogenesis of these infections is not well understood and research in this domain would benefit from the availability of a quantitative technique to determine viral load in clinical specimens. This study describes the development of a real-time RT-qPCR assay using hydrolysis TaqMan probe and a competitive RNA internal control. The assay has high specificity and can be used for a large sample of distinct enterovirus strains and serotypes. The reproducible limit of detection was estimated at 1875 copies/ml of quantitative standards composed of RNA transcripts obtained from a cloned echovirus 30 genome. Technical performance was unaffected by the introduction of a competitive RNA internal control before RNA extraction. The mean enterovirus RNA concentration in an evaluation series of 15 archived cerebrospinal fluid specimens was determined at 4.78 log(10)copies/ml for the overall sample. The sensitivity and reproducibility of the real time RT-qPCR assay used in combination with the internal control to monitor the overall specimen process make it a valuable tool with applied research into enterovirus infections.
Smith, William L.; Chadwick, Sean G.; Toner, Geoffrey; Mordechai, Eli; Adelson, Martin E.; Aguin, Tina J.; Sobel, Jack D.
Bacterial vaginosis (BV) is the most common gynecological infection in the United States. Diagnosis based on Amsel's criteria can be challenging and can be aided by laboratory-based testing. A standard method for diagnosis in research studies is enumeration of bacterial morphotypes of a Gram-stained vaginal smear (i.e., Nugent scoring). However, this technique is subjective, requires specialized training, and is not widely available. Therefore, a highly accurate molecular assay for the diagnosis of BV would be of great utility. We analyzed 385 vaginal specimens collected prospectively from subjects who were evaluated for BV by clinical signs and Nugent scoring. We analyzed quantitative real-time PCR (qPCR) assays on DNA extracted from these specimens to quantify nine organisms associated with vaginal health or disease: Gardnerella vaginalis, Atopobium vaginae, BV-associated bacteria 2 (BVAB2, an uncultured member of the order Clostridiales), Megasphaera phylotype 1 or 2, Lactobacillus iners, Lactobacillus crispatus, Lactobacillus gasseri, and Lactobacillus jensenii. We generated a logistic regression model that identified G. vaginalis, A. vaginae, and Megasphaera phylotypes 1 and 2 as the organisms for which quantification provided the most accurate diagnosis of symptomatic BV, as defined by Amsel's criteria and Nugent scoring, with 92% sensitivity, 95% specificity, 94% positive predictive value, and 94% negative predictive value. The inclusion of Lactobacillus spp. did not contribute sufficiently to the quantitative model for symptomatic BV detection. This molecular assay is a highly accurate laboratory tool to assist in the diagnosis of symptomatic BV. PMID:26818677
Faye, Martin; Dacheux, Laurent; Weidmann, Manfred; Diop, Sylvie Audrey; Loucoubar, Cheikh; Bourhy, Hervé; Sall, Amadou Alpha; Faye, Ousmane
Rabies virus (RABV) remains one of the most important global zoonotic pathogens. RABV causes rabies, an acute encephalomyelitis associated with a high rate of mortality in humans and animals and affecting different parts of the world, particularly in Asia and Africa. Confirmation of rabies diagnosis relies on laboratory diagnosis, in which molecular techniques such as detection of viral RNA by reverse transcription polymerase chain reaction (RT-PCR) are increasingly being used. In this study, two real-time quantitative RT-PCR assays were developed for large-spectrum detection of RABV, with a focus on African isolates. The primer and probe sets were targeted highly conserved regions of the nucleoprotein (N) and polymerase (L) genes. The results indicated the absence of non-specific amplification and cross-reaction with a range of other viruses belonging to the same taxonomic family, i.e. Rhabdoviridae, as well as negative brain tissues from various host species. Analytical sensitivity ranged between 100 to 10 standard RNA copies detected per reaction for N-gene and L-gene assays, respectively. Effective detection and high sensitivity of these assays on African isolates showed that they can be successfully applied in general research and used in diagnostic process and epizootic surveillance in Africa using a double-check strategy. Copyright © 2017 Elsevier B.V. All rights reserved.
Kanthaswamy, S; Premasuthan, A; Ng, J; Satkoski, J; Goyal, V
In the United States, human forensic evidence collected from crime scenes is usually comingled with biomaterial of canine and feline origins. Knowledge of the concentration of nuclear DNA extracted from a crime scene biological sample and the species from which the sample originated is essential for DNA profiling. The ability to accurately detect and quantify target DNA in mixed-species samples is crucial when target DNA may be overwhelmed by non-target DNA. We have designed and evaluated a species-specific (human, dog and cat) nuclear DNA identification assay based on the TaqMan(®) quantitative real-time PCR (qPCR) technology that can simultaneously detect and measure minute quantities of DNA specific to either humans, dogs and/or cats. The fluorogenic triplex assay employs primers and hydrolysis probes that target the human TH01 locus as well as the dog and cat Melanocortin 1 Receptor (MC1R) sequences in a species-specific manner. We also demonstrate that the assay is a highly sensitive, reliable and robust method for identifying and quantifying mixed-species templates of human-dog-cat origin with as little as 0.4 pg of human and cat nuclear DNA, respectively, and 4.0 pg of dog nuclear DNA.
Allender, Matthew C; Bunick, David; Dzhaman, Elena; Burrus, Lucienne; Maddox, Carol
Fungal pathogens threatening the conservation of wildlife are becoming increasingly common. Since 2008, free-ranging snakes across North America have been experiencing a marked increase in the prevalence of snake fungal disease associated with Ophidiomyces ophiodiicola. Diagnosis has historically relied on histology, microbiology, and conventional polymerase chain reaction (PCR). More sensitive methods are needed to adequately characterize the epidemiology. The current study describes the development of a real-time PCR (qPCR) assay for detecting a segment of the internal transcribed spacer 1 region between the 18S and 5.8S ribosomal RNA gene. The assay was able to detect as few as 1.05 × 10(1) gene copies per reaction. An additional 4 positive cases were detected when comparing a conventional PCR (n = 3) and the qPCR (n = 7) when used on swab samples from 47 eastern massasauga rattlesnakes. The newly developed assay is a sensitive and specific tool for surveillance and monitoring in the conservation of free-ranging snakes.
Rahman, Md Mahfujur; Hamid, Sharifah Bee Abd; Basirun, Wan Jefrey; Bhassu, Subha; Rashid, Nur Raifana Abdul; Mustafa, Shuhaimi; Mohd Desa, Mohd Nasir; Ali, Md Eaqub
This paper describes a short-amplicon-based TaqMan probe quantitative real-time PCR (qPCR) assay for the quantitative detection of canine meat in chicken nuggets, which are very popular across the world, including Malaysia. The assay targeted a 100-bp fragment of canine cytb gene using a canine-specific primer and TaqMan probe. Specificity against 10 different animals and plants species demonstrated threshold cycles (Ct) of 16.13 ± 0.12 to 16.25 ± 0.23 for canine DNA and negative results for the others in a 40-cycle reaction. The assay was tested for the quantification of up to 0.01% canine meat in deliberately spiked chicken nuggets with 99.7% PCR efficiency and 0.995 correlation coefficient. The analysis of the actual and qPCR predicted values showed a high recovery rate (from 87% ± 28% to 112% ± 19%) with a linear regression close to unity (R(2) = 0.999). Finally, samples of three halal-branded commercial chicken nuggets collected from different Malaysian outlets were screened for canine meat, but no contamination was demonstrated.
Kolb, Steffen; Knief, Claudia; Stubner, Stephan; Conrad, Ralf
Methane oxidation in soils is mostly accomplished by methanotrophic bacteria. Little is known about the abundance of methanotrophs in soils, since quantification by cultivation and microscopic techniques is cumbersome. Comparison of 16S ribosomal DNA and pmoA (alpha subunit of the particulate methane monooxygenase) phylogenetic trees showed good correlation and revealed five distinct groups of methanotrophs within the alpha and gamma subclasses of Proteobacteria: the Methylococcus group, the Methylobacter/Methylosarcina group, the Methylosinus group, the Methylocapsa group, and the forest clones group (a cluster of pmoA sequences retrieved from forest soils). We developed quantitative real-time PCR assays with SybrGreen for each of these five groups and for all methanotrophic bacteria by targeting the pmoA gene. Detection limits were between 10(1) and 10(2) target molecules per reaction for all assays. Real-time PCR analysis of soil samples spiked with cells of Methylococcus capsulatus, Methylomicrobium album, and Methylosinus trichosporium recovered almost all the added bacteria. Only the Methylosinus-specific assay recovered only 20% of added cells, possibly due to a lower lysis efficiency of type II methanotrophs. Analysis of the methanotrophic community structure in a flooded rice field soil showed (5.0 +/- 1.4) x 10(6) pmoA molecules g(-1) for all methanotrophs. The Methylosinus group was predominant (2.7 x 10(6) +/- 1.1 x 10(6) target molecules g(-1)). In addition, bacteria of the Methylobacter/Methylosarcina group were abundant (2.0 x 10(6) +/- 0.9 x 10(6) target molecules g of soil(-1)). On the other hand, pmoA affiliated with the forest clones and the Methylocapsa group was below the detection limit of 1.9 x 10(4) target molecules g of soil(-1). Our results showed that pmoA-targeted real-time PCR allowed fast and sensitive quantification of the five major groups of methanotrophs in soil. This approach will thus be useful for quantitative analysis of the
Newby, Deborah Trishelle; Hadfield, Ted; Roberto, Francisco Figueroa
Real-time PCR provides a means of detecting and quantifying DNA targets by monitoring PCR product accumulation during cycling as indicated by increased fluorescence. A number of different approaches can be used to generate the fluorescence signal. Three approaches—SYBR Green I (a double-stranded DNA intercalating dye), 5'-exonuclease (enzymatically released fluors), and hybridization probes (fluorescence resonance energy transfer)—were evaluated for use in a real-time PCR assay to detect Brucella abortus. The three assays utilized the same amplification primers to produce an identical amplicon. This amplicon spans a region of the B. abortus genome that includes portions of the alkB gene and the IS711 insertion element. All three assays were of comparable sensitivity, providing a linear assay over 7 orders of magnitude (from 7.5 ng down to 7.5 fg). However, the greatest specificity was achieved with the hybridization probe assay.
Park, Y; Kim, Y S; Kim, S I; Kim, H; Kim, H S
The human leukocyte antigen (HLA)-B*51 genotype is one of the well-known genetic factors associated with the development of Behcet's disease. We evaluated three sequence-specific priming (SSP) assays and one real-time PCR assay for detecting HLA-B*51 alleles using 93 whole blood samples, which were genotyped by high-resolution sequence-based typing (SBT). All HLA-B*51 alleles determined by SBT were detected by the four evaluated assays, and the results for all HLA-B alleles other than HLA-B*51 were negative on all assays. Thus, all HLA-B51 tests showed 100% sensitivity and 100% specificity for detecting HLA-B*51 alleles. The three SSP assays and the real-time PCR test for HLA-B*51 genotyping are simple, but reliable for detecting HLA-B*51 alleles in clinical laboratories.
Robers, Matthew B; Binkowski, Brock F; Cong, Mei; Zimprich, Chad; Corona, Cesear; McDougall, Mark; Otto, George; Eggers, Christopher T; Hartnett, Jim; Machleidt, Thomas; Fan, Frank; Wood, Keith V
Ligand-mediated endocytosis is a key autoregulatory mechanism governing the duration and intensity of signals emanating from cell surface receptors. Due to the mechanistic complexity of endocytosis and its emerging relevance in disease, simple methods capable of tracking this dynamic process in cells have become increasingly desirable. We have developed a bioluminescent reporter technology for real-time analysis of ligand-mediated receptor endocytosis using genetic fusions of NanoLuc luciferase with various G-protein-coupled receptors (GPCRs). This method is compatible with standard microplate formats, which should decrease work flows for high-throughput screens. This article also describes the application of this technology to endocytosis of epidermal growth factor receptor (EGFR), demonstrating potential applicability of the method beyond GPCRs.
Jung, Yu Jung; Kim, Ji-Youn; Song, Dong Joon; Koh, Won-Jung; Huh, Hee Jae; Ki, Chang-Seok; Lee, Nam Yong
We evaluated the analytical performance of M. tuberculosis complex (MTBC)/nontuberculous mycobacteria (NTM) PCR assays for differential identification of MTBC and NTM using culture-positive liquid media. Eighty-five type strains and 100 consecutive mycobacterial liquid media cultures (MGIT 960 system) were analyzed by a conventional PCR assay (MTB-ID(®) V3) and three real-time PCR assays (AdvanSure™ TB/NTM real-time PCR, AdvanSure; GENEDIA(®) MTB/NTM Detection Kit, Genedia; Real-Q MTB & NTM kit, Real-Q). The accuracy rates for reference strains were 89.4%, 100%, 98.8%, and 98.8% for the MTB-ID V3, AdvanSure, Genedia, and Real-Q assays, respectively. Cross-reactivity in the MTB-ID V3 assay was mainly attributable to non-mycobacterium Corynebacterineae species. The diagnostic performance was determined using clinical isolates grown in liquid media, and the overall sensitivities for all PCR assays were higher than 95%. In conclusion, the three real-time PCR assays showed better performance in discriminating mycobacterium species and non-mycobacterium Corynebacterineae species than the conventional PCR assay.
Yang, Yang; Qin, Xiaodong; Song, Yiming; Zhang, Wei; Hu, Gaowei; Dou, Yongxi; Li, Yanmin; Zhang, Zhidong
Peste des petits ruminants (PPR) is an economically important, Office International des Epizooties (OIE) notifiable, transboundary viral disease of small ruminants such as sheep and goat. PPR virus (PPRV), a negative-sense single-stranded RNA virus, is the causal agent of PPR. Therefore, sensitive, specific and rapid diagnostic assay for the detection of PPRV are necessary to accurately and promptly diagnose suspected case of PPR. In this study, reverse transcription recombinase polymerase amplification assays using real-time fluorescent detection (real-time RT-RPA assay) and lateral flow strip detection (LFS RT-RPA assay) were developed targeting the N gene of PPRV. The sensitivity of the developed real-time RT-RPA assay was as low as 100 copies per reaction within 7 min at 40 °C with 95% reliability; while the sensitivity of the developed LFS RT-RPA assay was as low as 150 copies per reaction at 39 °C in less than 25 min. In both assays, there were no cross-reactions with sheep and goat pox viruses, foot-and-mouth disease virus and Orf virus. These features make RPA assay promising candidates either in field use or as a point of care diagnostic technique.
Eischeid, Anne C; Kasko, Sasha M
Real-time PCR has been used widely in numerous fields. In food safety, it has been applied to detection of microbes and other contaminants, including food allergens. Interest in rapid (fast) cycling real-time PCR has grown because it yields results in less time than does conventional cycling. However, fast cycling can adversely affect assay performance. Here we report on tests of commercial master mixes specifically designed for fast real-time PCR using a shrimp allergen assay we previously developed and validated. The objective of this work was to determine whether specialized commercial master mixes lead to improved assay performance in rapid cycling. Real-time PCR assays were carried out using four different master mixes and two different rapid cycling protocols. Results indicated that specialized master mixes did yield quality results. In many cases, linear ranges spanned up to 7 orders of magnitude, R(2) values were at least 0.95, and reaction efficiencies were within or near the optimal range of 90 to 110%. In the faster of the two rapid cycling protocols tested, assay performance and PCR amplification were markedly better for the shorter PCR product. In conclusion, specialized commercial master mixes were effective as part of rapid cycling protocols, but conventional cycling as used in our previous work is more reliable for the shrimp assay tested.
Partial comparison of the NxTAG Respiratory Pathogen Panel Assay with the Luminex xTAG Respiratory Panel Fast Assay V2 and singleplex real-time polymerase chain reaction for detection of respiratory pathogens.
Esposito, Susanna; Scala, Alessia; Bianchini, Sonia; Presicce, Maria Lory; Mori, Alessandro; Sciarrabba, Calogero Sathya; Fior, Giulia; Principi, Nicola
In this study, 185 nasopharyngeal swabs were tested to compare the sensitivity and specificity of the Luminex NxTAG (NxTAG) Respiratory Pathogen Panel (RPP) Assay with those of the Luminex Respiratory Virus Panel (RVP) Fast Assay v2 and singleplex real-time polymerase chain reaction (PCR). The NxTAG Assay identified at least one infectious agent in 164 (88.7%) of the swabs. In 91 (6.2%) tests with negative results with the RVP Fast Assay v2, a virus was identified by the NxTAG (P < 0.001). With the NxTAG Assay, the detection rates were significantly higher for respiratory syncytial virus (P = 0.003), human metapneumovirus (P < 0.001), human rhinovirus/human enterovirus (P = 0.009) and human adenovirus (P < 0.001). Finally, the NxTAG Assay identified M. pneumoniae in 32 of 44 (72.7%) PCR-positive samples. However, the concordance with real-time PCR results was low for both assays. In conclusion, the results indicate that the NxTAG Assay overcomes some of the limitations of previous Luminex assays, although further studies are needed for a more complete evaluation of the new assay.
Fu, Qiang; Tang, Jun; Cui, Meng; Zheng, Zhong; Liu, Zhiqiang; Liu, Shuying
The continuous enzymatic assay based on ESI-MS was developed to real-time monitoring of enzymatic reactions of acetylcholinesterase (AChE). The changes of product concentrations were continuously measured. Calibration curves were established for quantitative calculation. By this method, the Michaelis constant (Km) of acetylcholinesterase was determined to be 70.60±0.93μM and Huperzine A as an effective inhibitor of acetylcholinesterase displayed a mixed inhibition with competitive and noncompetitive inhibition behaviors. The half maximal inhibitory concentration (IC50) and inhibition constant (Ki) value of Huperzine A were also calculated as 48.51±1.16nM and 26.73±0.27nM, respectively. This method provides the rapid and accurate ways to monitor enzyme reactions.
Boppana, Suresh B.; Ross, Shannon A.; Novak, Zdenek; Shimamura, Masako; Tolan, Robert W.; Palmer, April L.; Ahmed, Amina; Michaels, Marian G.; Sánchez, Pablo J.; Bernstein, David I.; Britt, William J.; Fowler, Karen B.
Context Reliable methods to screen newborns for congenital cytomegalovirus (CMV) infection are needed for identification of infants at increased risk for hearing loss. Since dried blood spots (DBS) are routinely collected for metabolic screening from all newborns in the United States, there has been interest in using DBS polymerase chain reaction (PCR)-based methods for newborn CMV screening. Objective To determine the diagnostic accuracy of DBS real-time PCR assays for newborn CMV screening Design, Setting, and Participants Between March 2007 and May 2008, infants born at seven medical centers in the U.S. were enrolled in the CMV and Hearing Multicenter Screening (CHIMES) study. Newborn saliva specimens were tested for the detection of early antigen fluorescent foci (DEAFF). Results of saliva DEAFF were compared with a single-primer (from 03/07 to 12/07) and a two-primer (from 01/08 to 05/08) DBS real-time PCR. Infants positive by screening DEAFF or PCR were enrolled in follow-up to confirm congenital infection by the reference standard method, DEAFF on saliva or urine. Main Outcome Measures Sensitivity, specificity and positive and negative likelihood ratios (LRs) of single-primer and two-primer DBS real-time PCR assays for identifying infants with confirmed congenital CMV infection. Results Congenital CMV infection was confirmed in 92 of 20,448 (0.45%; 95% CI, 0.36–0.55) infants. Ninety-one of 92 infants were saliva DEAFF positive on screening. Of the 11,422 infants screened using the single-primer DBS PCR, 17 of 60 (28%) infants were positive with this assay, whereas, among the 9,026 infants screened using the two-primer DBS PCR, 11 of 32 (34%) infants were positive. The single-primer DBS PCR identified congenital CMV infection with a sensitivity of 28.3% (95% CI, 17.4–41.4%), specificity, 99.9% (95% CI, 99.9–100%), positive LR, 803.7 (95% CI, 278.7–2317.9), and negative LR, 0.7 (95% CI, 0.6–0.8). The positive and negative predictive values of the
Wang, Xiaobo; Zhang, Zhiyang; Ma, Xiaoyan; Wen, Jinghan; Geng, Zhirong; Wang, Zhilin
An anthracene-armed tetraaza macrocyclic fluorescent probe 3-(9-anthrylmethyl)-3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene(l) for detecting Zn(2+) in aqueous medium was synthesized. L-Zn(2+) complex, showed selectivity toward pyrophosphate ion (PPi) by quenching the fluorescence in aqueous HEPES buffer (pH 7.4). Furthermore, L-Zn(2+) was also used to set up a real-time fluorescence assay for monitoring enzyme activities of alkaline phosphatase (ALP) and adenosine triphosphate sulfurylase (ATPS). In the presence of ALP inhibitor Na3VO4 and ATPS inhibitor chlorate, two enzymes activities decreased obviously, respectively.
Kumar, R Satish; Ramesh, S
Candida glabrata is an opportunistic human pathogen known to cause systemic and vaginal candidiasis. Rapid detection of Candida glabrata is indispensable for appropriate selection of antifungal drugs for chemotherapy. The study describes a unique intein-containing DNA fragment for specific detection of C. glabrata. The designed oligonucleotides detected C. glabrata (Ct mean: 24.75 ± 1.1 and Tm: 70.08 ± 0.23°C) in Real-Time PCR assays. The fluorescent signals were negative when the primers were tested for cross-species and cross-genera amplifications. In conclusion, our study recommends a novel primer set for developing a quick identification system which does not require laborious and time-consuming experimentations.
Wang, Yuexia; Yang, Ming; Liu, Shuchun; Chen, Wanyi; Suo, Biao
Real-time polymerase chain reaction (PCR) allows rapid detection of Salmonella in frozen dairy products, but it might cause a false positive detection result because it might amplify DNA from dead target cells as well. In this study, Salmonella-free frozen ice cream was initially inoculated with heat-killed Salmonella Typhimurium cells and stored at -18°C. Bacterial DNA extracted from the sample was amplified using TaqMan probe-based real-time PCR targeting the invA gene. Our results indicated that DNA from the dead cells remained stable in frozen ice cream for at least 20 days, and could produce fluorescence signal for real-time PCR as well. To overcome this limitation, propidium monoazide (PMA) was combined with real-time PCR. PMA treatment can effectively prevent PCR amplification from heat-killed Salmonella cells in frozen ice cream. The PMA real-time PCR assay can selectively detect viable Salmonella at as low as 10(3) CFU/mL. Combining 18 hours of pre-enrichment with the assay allows for the detection of viable Salmonella at 10(0) CFU/mL and avoiding the false-positive result of dead cells. The PMA real-time PCR assay provides an alternative specifically for detection of viable Salmonella in ice cream. However, when the PMA real-time PCR assay was evaluated in ice cream subjected to frozen storage, it obviously underestimated the contamination situation of viable Salmonella, which might lead to a false negative result. According to this result, the use of enrichment prior to PMA real-time PCR analysis remains as the more appropriate approach. Copyright © 2015. Published by Elsevier B.V.
Shallom, Shamira J.; Moura, Natalia S.; Olivier, Kenneth N.; Sampaio, Elizabeth P.; Holland, Steven M.
Members of the Mycobacterium abscessus group (MAG) cause lung, soft tissue, and disseminated infections. The oral macrolides clarithromycin and azithromycin are commonly used for treatment. MAG can display clarithromycin resistance through the inducible erm(41) gene or via acquired mutations in the rrl (23S rRNA) gene. Strains harboring a truncation or a T28C substitution in erm(41) lose the inducible resistance trait. Phenotypic detection of clarithromycin resistance requires extended incubation (14 days), highlighting the need for faster methods to detect resistance. Two real-time PCR-based assays were developed to assess inducible and acquired clarithromycin resistance and tested on a total of 90 clinical and reference strains. A SYBR green assay was designed to distinguish between a full-length and truncated erm(41) gene by temperature shift in melting curve analysis. Single nucleotide polymorphism (SNP) allele discrimination assays were developed to distinguish T or C at position 28 of erm(41) and 23S rRNA rrl gene mutations at position 2058 and/or 2059. Truncated and full-size erm(41) genes were detected in 21/90 and 69/90 strains, respectively, with 64/69 displaying T at nucleotide position 28 and 5/69 containing C at that position. Fifteen isolates showed rrl mutations conferring clarithromycin resistance, including A2058G (11 isolates), A2058C (3 isolates), and A2059G (1 isolate). Targeted sequencing and phenotypic assessment of resistance concurred with molecular assay results. Interestingly, we also noted cooccurring strains harboring an active erm(41), inactive erm(41), and/or acquired mutational resistance, as well as slowly growing MAG strains and also strains displaying an inducible resistance phenotype within 5 days, long before the recommended 14-day extended incubation. PMID:26269619
Clayton, Evelyn M.; Hill, Colin; Cotter, Paul D.; Ross, R. Paul
Due to the severity of the food-borne infection listeriosis, strict legislation governs the detectable and permissible limits at which Listeria monocytogenes is permitted in foods. These requirements, coupled with the ubiquitous nature of L. monocytogenes strains and the potential for epidemic outbreaks, mean that the pathogen can devastate affected sectors of the food industry. Although almost all L. monocytogenes strains have the potential to cause listeriosis, those implicated in the vast majority of epidemics belong to a subset of strains belonging to evolutionary lineage I. It has been established that a significant proportion of these strains, including those implicated in the majority of outbreaks, produce an additional hemolysin, designated listeriolysin S (LLS), which may be responsible for the enhanced virulence of these strains. In order to ultimately establish this definitively, it is important to first be able to rapidly discriminate between LLS-positive and -negative strains. Here, after essential genes within the LLS-encoding cluster, Listeria pathogenicity island 3, were identified by deletion mutagenesis, a real-time PCR assay which targets one such gene, llsX, was developed as a means of identifying LLS-positive L. monocytogenes. The specificity of the assay was validated against a panel of 40 L. monocytogenes strains (20 of which were LLS positive) and 25 strains representative of other Listeria species. Furthermore, 1 CFU of an LLS-positive strain per 25 g/ml of spiked foods was detected in less than 30 h when the assay was coupled with culture enrichment. The detection limit of this assay was 10 genome equivalents. PMID:21075895
De Zoysa, Aruni; Efstratiou, Androulla; Mann, Ginder; Harrison, Timothy G; Fry, Norman K
Toxigenic corynebacteria are uncommon in the UK; however, laboratory confirmation by the national reference laboratory can inform public health action according to national guidelines. Standard phenotypic tests for identification and toxin expression of isolates can take from ≥24 to ≥48 h from receipt. To decrease the time to result, a real-time PCR (qPCR) assay was developed for confirmation of both identification of Corynebacterium diphtheriae and Corynebacterium ulcerans/Corynebacterium pseudotuberculosis and detection of the diphtheria toxin gene. Target genes were the RNA polymerase β-subunit-encoding gene (rpoB) and A-subunit of the diphtheria toxin gene (tox). Green fluorescent protein DNA (gfp) was used as an internal process control. qPCR results were obtained within 3 to 4 h after receipt of isolate. The assay was validated according to published guidelines and demonstrated high diagnostic sensitivity (100 %), high specificity (98-100 %) and positive and negative predictive values of 91 to 100 % and 100 %, respectively, compared to both block-based PCR and the Elek test, together with a greatly reduced time from isolate receipt to reporting. Limitations of the qPCR assay were the inability to distinguish between C. ulcerans and C. pseudotuberculosis and that the presence of the toxin gene as demonstrated by qPCR may not always predict toxin expression. Thus, confirmation of expression of diphtheria toxin is always sought using the phenotypic Elek test. The new qPCR assay was formally introduced as the front-line test for putative toxigenic corynebacteria to inform public health action in England and Wales on 1 April 2014.
Nordentoft, Steen; Kabell, Susanne; Pedersen, Karl
Infections caused by members of the Chlamydiaceae family have long been underestimated due to the requirement of special laboratory facilities for the detection of this group of intracellular pathogens. Furthermore, new studies of this group of intracellular pathogens have revealed that host specificity of different species is not as clear as recently believed. As most members of the genus Chlamydophila have shown to be transmissible from animals to humans, sensitive and fast detection methods are required. In this study, SYBR green-based real-time assays were developed that detect all members of Chlamydiaceae and differentiate the most prevalent veterinary Chlamydophila species: Cp. psittaci, Cp. abortus, Cp. felis, and Cp. caviae. By adding bovine serum albumin to the master mixes, target DNA could be detected directly in crude lysates of enzymatically digested conjunctival or pharyngeal swabs or tissue specimens from heart, liver, and spleen without further purification. The assays were evaluated on veterinary specimens where all samples were screened using a family-specific PCR, and positive samples were further tested using species-specific PCRs. Cp. psittaci was detected in 47 birds, Cp. felis was found in 10 cats, Cp. caviae was found in one guinea pig, and Cp. abortus was detected in one sheep. The screening assay appeared more sensitive than traditional microscopical examination of stained tissue smears. By combining a fast, robust, and cost-effective method for sample preparation with a highly sensitive family-specific PCR, we were able to screen for Chlamydiaceae in veterinary specimens and confirm the species in positive samples with additional PCR assays.
Walsh, Kenneth B
The cardiac acetylcholine-activated K(+) channel (I(K,Ach)) represents a novel target for drug therapy in the treatment of atrial fibrillation (AF). This channel is a member of the G-protein-coupled inward rectifier K(+) (GIRK) channel superfamily and is composed of the GIRK1/4 (Kir3.1 and Kir3.4) subunits. The goal of this study was to develop a cell-based screening assay for identifying new blockers of the GIRK1/4 channel. The mouse atrial HL-1 cell line, expressing the GIRK1/4 channel, was plated in 96-well plate format, loaded with the fluorescent membrane potential-sensitive dye bis-(1,3-dibutylbarbituric acid) trimethine oxonol (DiBAC(4)(3)) and measured using a fluorescent imaging plate reader (FLIPR). Application of the muscarinic agonist carbachol to the cells caused a rapid, time-dependent decrease in the fluorescent signal, indicative of K(+) efflux through the GIRK1/4 channel (carbachol vs. control solution, Z' factor = 0.5-0.6). The GIRK1/4 channel fluorescent signal was blocked by BaCl(2) and enhanced by increasing the driving force for K(+) across the cell membrane. To test the utility of the assay for screening GIRK1/4 channel blockers, cells were treated with a small compound library of Na(+) and K(+) channel modulators. Analogues of amiloride and propafenone were identified as channel blockers at concentrations less than 1 µM. Thus, the GIRK1/4 channel assay may be used in the development of new and selective agents for treating AF.
Ibatullin, Farid M; Banasiak, Alicja; Baumann, Martin J; Greffe, Lionel; Takahashi, Junko; Mellerowicz, Ewa J; Brumer, Harry
There currently exists a diverse array of molecular probes for the in situ localization of polysaccharides, nucleic acids, and proteins in plant cells, including reporter enzyme strategies (e.g. protein-glucuronidase fusions). In contrast, however, there is a paucity of methods for the direct analysis of endogenous glycoside hydrolases and transglycosidases responsible for cell wall remodeling. To exemplify the potential of fluorogenic resorufin glycosides to address this issue, a resorufin beta-glycoside of a xylogluco-oligosaccharide (XXXG-beta-Res) was synthesized as a specific substrate for in planta analysis of XEH activity. The resorufin aglycone is particularly distinguished for high sensitivity in muro assays due to a low pK(a) (5.8) and large extinction coefficient (epsilon 62,000 M(-1) cm(-1)), long-wavelength fluorescence (excitation 571 nm/emission 585 nm), and high quantum yield (0.74) of the corresponding anion. In vitro analyses demonstrated that XXXG-beta-Res is hydrolyzed by the archetypal plant XEH, nasturtium (Tropaeolum majus) NXG1, with classical Michaelis-Menten substrate saturation kinetics and a linear dependence on both enzyme concentration and incubation time. Further, XEH activity could be visualized in real time by observing the localized increase in fluorescence in germinating nasturtium seeds and Arabidopsis (Arabidopsis thaliana) inflorescent stems by confocal microscopy. Importantly, this new in situ XEH assay provides an essential complement to the in situ xyloglucan endotransglycosylase assay, thus allowing delineation of the disparate activities encoded by xyloglucan endotransglycosylase/hydrolase genes directly in plant tissues. The observation that XXXG-beta-Res is also hydrolyzed by diverse microbial XEHs indicates that this substrate, and resorufin glycosides in general, may find broad applicability for the analysis of wall restructuring by polysaccharide hydrolases during morphogenesis and plant-microbe interactions.
Mirhendi, Hossein; Motamedi, Marjan; Makimura, Koichi; Satoh, Kazuo
Early differentiation of dermatophytosis from other cutaneous mycoses is essential to avoid inaccurate therapy. DNA-based techniques including real-time PCR have increasingly been considered for detection of fungal elements in clinical specimens. In this study, after partial sequence analysis of beta tubulin (BT2) gene in 13 common and rare pathogenic dermatophyte species, a pan-dermatophyte primer and probe set was designed in a TaqMan probe-based PCR format. The sensitivity and specificity of the system was tested with 22 reference strains of dermatophytes, 234 positive clinical specimens, 32 DNA samples extracted from normal nails, several fungi other than dermatophytes and human DNAs. Analytical detection limit of the designed PCR on serially diluted DNAs of prepared recombinant plasmid indicated that only five molecules per sample are the minimum number for reliable detection by the assay. A total of 226 out of 234 (96.5%) DNAs extracted from clinical samples, but none of the 32 nail samples, from healthy volunteers were positive in PCR. The real-time PCR targeted beta tubulin gene established in this study could be a sensitive diagnostic tool which is significantly faster than the conventional culture method and should be useful in the clinical settings, in large-scale epidemiological studies and in clinical trials of antifungal therapy.
Alonso, José L; Amorós, Inmaculada; Cañigral, Irene
Cryptosporidium and Giardia are major causes of diarrheal disease in humans worldwide and are major causes of protozoan waterborne diseases. Two DNA TaqMan PCR-based Giardia and Cryptosporidium methods targeting a 74-bp sequence of the β-giardin Giardia gene and a 151-bp sequence of the COWP Cryptosporidium gene, respectively, were used as models to compare two different LNA/DNA TaqMan probes to improve the detection limit in a real-time PCR assay. The LNA probes were the most sensitive resulting in 0.96 to 1.57 lower C t values than a DNA Giardia TaqMan probe and 0.56 to 2.21 lower than a DNA Cryptosporidium TaqMan probe. Evaluation of TaqMan Giardia and Cryptosporidium probes with LNA substitutions resulted in real-time PCR curves with an earlier C t values than conventional DNA TaqMan probes. In conclusion, the LNA probes could be useful for more sensitive detection limits.
Buitrago, Dolores; Rocha, Ana; Tena-Tomás, Cristina; Vigo, Marta; Agüero, Montserrat; Jiménez-Clavero, Miguel Angel
In September 2010, an outbreak of disease in 2 wild bird species (red-legged partridge, Alectoris rufa; ring-necked pheasant, Phasianus colchicus) occurred in southern Spain. Bagaza virus (BAGV) was identified as the etiological agent of the outbreak. BAGV had only been reported before in Western Africa (Central African Republic, Senegal) and in India. The first occurrence of BAGV in Spain stimulated a demand for rapid, reliable, and efficacious diagnostic methods to facilitate the surveillance of this disease in the field. This report describes a real-time reverse transcription polymerase chain reaction (RT-PCR) method based on a commercial 5'-Taq nuclease-3' minor groove binder DNA probe and primers targeting the Bagaza NS5 gene. The method allowed the detection of BAGV with a high sensitivity, whereas other closely related flaviviruses (Usutu virus, West Nile virus, and Japanese encephalitis virus) were not detected. The assay was evaluated using field samples of red-legged partridges dead during the outbreak (n = 11), as well as samples collected from partridges during surveillance programs (n = 81). The results were compared to those obtained with a pan-flaviviral hemi-nested RT-PCR followed by nucleotide sequencing, which was employed originally to identify the virus involved in the outbreak. The results obtained with both techniques were 100% matching, indicating that the newly developed real-time RT-PCR is a valid technique for BAGV genome detection, useful in both diagnosis and surveillance studies.
Tannous, Joanna; Atoui, Ali; El Khoury, André; Kantar, Sally; Chdid, Nader; Oswald, Isabelle P; Puel, Olivier; Lteif, Roger
Due to the occurrence and spread of the fungal contaminants in food and the difficulties to remove their resulting mycotoxins, rapid and accurate methods are needed for early detection of these mycotoxigenic fungi. The polymerase chain reaction and the real time PCR have been widely used for this purpose. Apples are suitable substrates for fungal colonization mostly caused by Penicillium expansum, which produces the mycotoxin patulin during fruit infection. This study describes the development of a real-time PCR assay incorporating an internal amplification control (IAC) to specifically detect and quantify P. expansum. A specific primer pair was designed from the patF gene, involved in patulin biosynthesis. The selected primer set showed a high specificity for P. expansum and was successfully employed in a standardized real-time PCR for the direct quantification of this fungus in apples. Using the developed system, twenty eight apples were analyzed for their DNA content. Apples were also analyzed for patulin content by HPLC. Interestingly, a positive correlation (R(2) = 0.701) was found between P. expansum DNA content and patulin concentration. This work offers an alternative to conventional methods of patulin quantification and mycological detection of P. expansum and could be very useful for the screening of patulin in fruits through the application of industrial quality control. Copyright © 2015 Elsevier Ltd. All rights reserved.
Menard, J-P; Mazouni, C; Fenollar, F; Raoult, D; Boubli, L; Bretelle, F
The purpose of this investigation was to determine the diagnostic accuracy of quantitative real-time polymerase chain reaction (PCR) assay in diagnosing bacterial vaginosis versus the standard methods, the Amsel criteria and the Nugent score. The Amsel criteria, the Nugent score, and results from the molecular tool were obtained independently from vaginal samples of 163 pregnant women who reported abnormal vaginal symptoms before 20 weeks gestation. To determine the performance of the molecular tool, we calculated the kappa value, sensitivity, specificity, and positive and negative predictive values. Either or both of the Amsel criteria (≥3 criteria) and the Nugent score (score ≥7) indicated that 25 women (15%) had bacterial vaginosis, and the remaining 138 women did not. DNA levels of Gardnerella vaginalis or Atopobium vaginae exceeded 10(9) copies/mL or 10(8) copies/mL, respectively, in 34 (21%) of the 163 samples. Complete agreement between both reference methods and high concentrations of G. vaginalis and A. vaginae was found in 94.5% of women (154/163 samples, kappa value = 0.81, 95% confidence interval 0.70-0.81). The nine samples with discordant results were categorized as intermediate flora by the Nugent score. The molecular tool predicted bacterial vaginosis with a sensitivity of 100%, a specificity of 93%, a positive predictive value of 73%, and a negative predictive value of 100%. The quantitative real-time PCR assay shows excellent agreement with the results of both reference methods for the diagnosis of bacterial vaginosis.
Molina, Elena; Elguezabal, Natalia; Pérez, Valentín; Garrido, Joseba M.
Mycobacterium tuberculosis complex, Mycobacterium avium, and many other nontuberculous mycobacteria are worldwide distributed microorganisms of major medical and veterinary importance. Considering the growing epidemiologic significance of wildlife-livestock-human interrelation, developing rapid detection tools of high specificity and sensitivity is vital to assess their presence and accelerate the process of diagnosing mycobacteriosis. Here we describe the development and evaluation of a novel tetraplex real-time PCR for simultaneous detection of Mycobacterium genus, M. avium subspecies, and M. tuberculosis complex in an internally monitored single assay. The method was evaluated using DNA from mycobacterial (n = 38) and nonmycobacterial (n = 28) strains, tissues spiked with different CFU amounts of three mycobacterial species (n = 57), archival clinical samples (n = 233), and strains isolated from various hosts (n = 147). The minimum detectable DNA amount per reaction was 50 fg for M. bovis BCG and M. kansasii and 5 fg for M. avium subsp. hominissuis. When spiked samples were analyzed, the method consistently detected as few as 100 to 1,000 mycobacterial CFU per gram. The sensitivity and specificity values for the panel of clinical samples were 97.5 and 100% using a verified culture-based method as the reference method. The assays performed on clinical isolates confirmed these results. This PCR was able to identify M. avium and M. tuberculosis complex in the same sample in one reaction. In conclusion, the tetraplex real-time PCR we designed represents a highly specific and sensitive tool for the detection and identification of mycobacteria in routine laboratory diagnosis with potential additional uses. PMID:25588660
Sevilla, Iker A; Molina, Elena; Elguezabal, Natalia; Pérez, Valentín; Garrido, Joseba M; Juste, Ramón A
Mycobacterium tuberculosis complex, Mycobacterium avium, and many other nontuberculous mycobacteria are worldwide distributed microorganisms of major medical and veterinary importance. Considering the growing epidemiologic significance of wildlife-livestock-human interrelation, developing rapid detection tools of high specificity and sensitivity is vital to assess their presence and accelerate the process of diagnosing mycobacteriosis. Here we describe the development and evaluation of a novel tetraplex real-time PCR for simultaneous detection of Mycobacterium genus, M. avium subspecies, and M. tuberculosis complex in an internally monitored single assay. The method was evaluated using DNA from mycobacterial (n = 38) and nonmycobacterial (n = 28) strains, tissues spiked with different CFU amounts of three mycobacterial species (n = 57), archival clinical samples (n = 233), and strains isolated from various hosts (n = 147). The minimum detectable DNA amount per reaction was 50 fg for M. bovis BCG and M. kansasii and 5 fg for M. avium subsp. hominissuis. When spiked samples were analyzed, the method consistently detected as few as 100 to 1,000 mycobacterial CFU per gram. The sensitivity and specificity values for the panel of clinical samples were 97.5 and 100% using a verified culture-based method as the reference method. The assays performed on clinical isolates confirmed these results. This PCR was able to identify M. avium and M. tuberculosis complex in the same sample in one reaction. In conclusion, the tetraplex real-time PCR we designed represents a highly specific and sensitive tool for the detection and identification of mycobacteria in routine laboratory diagnosis with potential additional uses.
Hemarajata, P.; Yang, S.; Soge, O. O.; Klausner, J. D.
In the United States, 19.2% of Neisseria gonorrhoeae isolates are resistant to ciprofloxacin. We evaluated a real-time PCR assay to predict ciprofloxacin susceptibility using residual DNA from the Roche Cobas 4800 CT/NG assay. The results of the assay were 100% concordant with agar dilution susceptibility test results for 100 clinical isolates. Among 76 clinical urine and swab specimens positive for N. gonorrhoeae by the Cobas assay, 71% could be genotyped. The test took 1.5 h to perform, allowing the physician to receive results in time to make informed clinical decisions. PMID:26739156
Sales, Mariana L.; Fonseca, Antônio Augusto; Orzil, Lívia; Alencar, Andrea Padilha; Silva, Marcio Roberto; Issa, Marina Azevedo; Filho, Paulo Martins Soares; Lage, Andrey Pereira; Heinemann, Marcos Bryan
Mycobacterium tuberculosis is the major cause of tuberculosis in humans. This bacillus gained prominence with the occurrence of HIV, presenting itself as an important opportunistic infection associated with acquired immunodeficiency syndrome (AIDS). The current study aimed to develop a real-time PCR using Eva Green technology for molecular identification of M. tuberculosis isolates. The primers were designed to Rv1510 gene. Ninety nine samples of M. tuberculosis and sixty samples of M. bovis were tested and no sample of the bovine bacillus was detected by the qPCR. Statistical tests showed no difference between the qPCR and biochemical tests used to identify the Mycobacterium tuberculosis. The correlation between tests was perfect with Kappa index of 1.0 (p < 0.001, CI = 0.84 – 1.0). The diagnostic sensitivity and specificity were 100% (CI = 95.94% – 100%) and 100% (CI = 93.98% – 100%). This qPCR was developed with the goal of diagnosing the bacillus M. tuberculosis in samples of bacterial suspension. TB reference laboratories (health and agriculture sectors), public health programs and epidemiological studies probably may benefit from such method. PMID:25763042
Pereira, Mariana R; Rocha-Silva, Fabiana; Graciele-Melo, Cidiane; Lafuente, Camila R; Magalhães, Telcia; Caligiorne, Rachel B
The diagnosis of visceral leishmaniasis (VL) is a challenging issue and several studies worldwide have evaluated the different tools to reach a diagnostic solution. The polymerase chain reaction (PCR) has proven to be effective in detecting the genome of Leishmania species in different biological samples. In this study, we compared the conventional PCR and real-time PCR using the Sybr Green system and their application in molecular diagnosis of visceral leishmaniasis in peripheral blood as a biological sample. The genus-specific conserved region of kinetoplast DNA (kDNA) was the target of amplification. We studied 30 samples from patients with suspect of visceral leishmaniasis who were treated by the Medical Clinic of Santa Casa de Belo Horizonte Hospital, Brazil. Among the samples studied, 19 had a confirmed diagnosis for VL by serology and/or by clinical findings. Among these 19 samples, 63% (n = 12) presented positive results for serology and 79% (n = 15) positive results in both PCR methodologies. This fact suggests that the PCR technique can assist in the diagnosis of visceral leishmaniasis in patients who do not have detectable antibodies by serology but can present the genome of the parasite circulating in whole blood. Also, it was possible to observe that there was conformity between the results of the techniques of cPCR and qPCR using the Sybr Green system in 100% of samples analyzed. These data suggest that both PCR techniques were equally effective for detection of the genome of the parasite in the patient's blood.
Sales, Mariana L; Fonseca Júnior, Antônio Augusto; Orzil, Lívia; Alencar, Andrea Padilha; Silva, Marcio Roberto; Issa, Marina Azevedo; Soares Filho, Paulo Martins; Lage, Andrey Pereira; Heinemann, Marcos Bryan
Mycobacterium tuberculosis is the major cause of tuberculosis in humans. This bacillus gained prominence with the occurrence of HIV, presenting itself as an important opportunistic infection associated with acquired immunodeficiency syndrome (AIDS). The current study aimed to develop a real-time PCR using Eva Green technology for molecular identification of M. tuberculosis isolates. The primers were designed to Rv1510 gene. Ninety nine samples of M. tuberculosis and sixty samples of M. bovis were tested and no sample of the bovine bacillus was detected by the qPCR. Statistical tests showed no difference between the qPCR and biochemical tests used to identify the Mycobacterium tuberculosis. The correlation between tests was perfect with Kappa index of 1.0 (p < 0.001, CI = 0.84 - 1.0). The diagnostic sensitivity and specificity were 100% (CI = 95.94% - 100%) and 100% (CI = 93.98% - 100%). This qPCR was developed with the goal of diagnosing the bacillus M. tuberculosis in samples of bacterial suspension. TB reference laboratories (health and agriculture sectors), public health programs and epidemiological studies probably may benefit from such method.
Kast, Christina; Roetschi, Alexandra
Occasionally, melissopalynological analysis reveals the presence of baker's yeast (Saccharomyces cerevisiae) in honey sediments. A field experiment reproducing a common spring bee feeding practice, using sugar paste containing baker's yeast, was performed to understand how S. cerevisiae are introduced into honey. Apart from classical microscopy, a real-time quantitative PCR (qPCR) system specific for S. cerevisiae was established for quantification of S. cerevisiae in honeys. Results showed that S. cerevisiae cells are stored in the honey of the brood combs and are also transferred into honey in the supers. The concentrations of S. cerevisiae were highest in honey of the brood frames immediately after the feeding and decreased over time to low concentrations at the end of the year. A high content of S. cerevisiae cells were also found in the honey from supers of the spring harvest. Observed S. cerevisiae cells were not able to multiply in a high-sugar environment, such as honey, and their viability decreased rapidly after addition to the honey. The screening of 200 Swiss honeys revealed the presence of S. cerevisiae in 4.5% of the samples, as determined by microscopy and qPCR. Finally, the method described here may indicate an unwanted sucrose addition to honey through bee-feeding.
Xia, Hongyan; Gravelsina, Sabine; Öhrmalm, Christina; Ottoson, Jakob; Blomberg, Jonas
The high sequence variation of RNA viruses necessitates use of degenerate primers and probes or multiple primers and probes in molecular diagnostic assays. We showed previously that PCR amplification in two rounds, first with long target-specific primers and then with short generic primers, followed by detection using long probes, can tolerate sequence variation. Here we demonstrate that long primers and probes of up to 56 nucleotides can also be applied in real-time PCR for the detection of norovirus genogroup II with improved sensitivity. Probe design (method of incorporating quenchers, use of Zen internal quencher or traditional quenchers) greatly affects the sensitivity of the real-time PCR assays.
Jennings, Cheryl; Johnson, Victoria A.; Coombs, Robert W.; McKinnon, John E.; Bremer, James W.; Cobb, Bryan R.; Cloherty, Gavin A.; Mellors, John W.; Ribaudo, Heather J.
Discrepancies between HIV-1 RNA results assayed by different FDA-approved platforms have been reported. Plasma samples collected from 332 randomly selected clinical trial participants during the second year of antiretroviral treatment were assayed with three FDA-approved platforms: UltraSensitive Roche Amplicor Monitor, v1.5 (Monitor), the Abbott RealTime HIV-1 test on the m2000 system (Abbott), and the Roche TaqMan HIV-1 test, v2.0 (TaqMan). Samples from 61 additional participants with confirmed HIV-1 RNA levels of >50 copies/ml during trial follow-up were also included. Endpoints were HIV-1 RNA quantification of ≤50 copies/ml versus >50 copies/ml at an individual-sample level (primary) and determination of confirmed virologic failure (VF) from longitudinal samples. A total of 389 participants had results obtained from all assays on at least one sample (median = 6). Proportions of results of >50 copies/ml were 19% (Monitor), 22% (TaqMan), and 25% (Abbott). Despite indication of strong agreement (Cohen's kappa, 0.76 to 0.82), Abbott was more likely to detect HIV-1 RNA levels of >50 copies/ml than Monitor (matched-pair odds ratio [mOR] = 4.2; modified Obuchowski P < 0.001) and TaqMan (mOR = 2.1; P < 0.001); TaqMan was more likely than Monitor (mOR = 2.6; P < 0.001). Despite strong agreement in classifying VF across assay comparisons (kappa, 0.75 to 0.92), at a 50-copies/ml threshold, differences in the probability of VF classification (in the same direction as primary) were apparent (all McNemar's P < 0.007). At a 200-copies/ml VF threshold, no differences between assays were apparent (all P > 0.13). Despite strong agreement among assays, significant differences were observed with respect to detecting HIV-1 RNA levels of >50 copies/ml and identifying VF at the 50-copies/ml threshold. This has important implications for the definition of VF in clinical trials and clinical practice. PMID:26063861
André, Murielle; Reghin, Sylviane; Boussard, Estelle; Lempereur, Laurent; Maisonneuve, Stéphane
Residual host cellular DNA (rcDNA) is one of the principal risk associated with continuous cell lines derived medicines such as viral vaccines. To assess rcDNA degradation, we suggest two quantitative real-time PCR assays designed to separately quantify target sequences shorter and longer than the 200 bp risk limit, the relative abundance of both targets reflecting the extent of rcDNA fragmentation. The conserved multicopy ribosomal 18S RNA gene was targeted to detect host cell templates from most mammalian cell substrates commonly used in the manufacture of human viral vaccines. The detection range of the method was assessed on purified DNA templates from different animal origins. The standard calibrator origin and structural conformation were shown crucial to achieve accurate quantification. Artificial mixtures of PCR products shorter and longer than 200 bp were used as a model to check the ability of the assay to estimate the fragment size distribution. The method was successfully applied to a panel of Vero cell derived vaccines and could be used as a universal method for determination of both content and size distribution of rcDNA in vaccines.
Niu, Xiaoyu; Chen, Hao; Yang, Jing; Yu, Xianglong; Ti, Jinfeng; Wang, Aihua; Diao, Youxiang
The newly emerged disease, duck beak atrophy and dwarfism syndrome (BADS), is caused by novel goose parovirus (N-GPV). Although N-GPV infection has severe consequences, few methods for detecting this virus have been developed. Therefore, the availability of rapid and reliable molecular diagnostic methods would aid future studies of this novel virus. Clinical specimens from 138 suspected cases of N-GPV infection and 120 cloacal swabs from breeding ducks were used in this study. The targeted sequence of N-GPV cloned into the pMD18-T vector was used to generate the N-GPV DNA standard curve. The specificity of the assay was validated using duck plague virus, GPV, duck hepatitis virus, avian influenza virus, duck reovirus, tembusu virus, and fowl adenovirus. The lowest limit of detection was 8.8×10(1)copies/μL with a good linear standard curve (Y=-3.3682X+37.220, R(2)=0.9953) over a wide range of input DNA, of which the concentration was between 8.8×10(1) to 8.8×10(8)copies/μL. The results show that the real-time PCR assay is a highly sensitive, specific, reproducible, and versatile method for quantitatively detecting N-GPV DNA, and thus can be used to detect this virus, thereby facilitating epidemiological investigations of animals with BADS.
Malnati, Mauro S; Scarlatti, Gabriella; Gatto, Francesca; Salvatori, Francesca; Cassina, Giulia; Rutigliano, Teresa; Volpi, Rosy; Lusso, Paolo
Quantification of human immunodeficiency virus type-1 (HIV-1) proviral DNA is increasingly used to measure the HIV-1 cellular reservoirs, a helpful marker to evaluate the efficacy of antiretroviral therapeutic regimens in HIV-1-infected individuals. Furthermore, the proviral DNA load represents a specific marker for the early diagnosis of perinatal HIV-1 infection and might be predictive of HIV-1 disease progression independently of plasma HIV-1 RNA levels and CD4(+) T-cell counts. The high degree of genetic variability of HIV-1 poses a serious challenge for the design of a universal quantitative assay capable of detecting all the genetic subtypes within the main (M) HIV-1 group with similar efficiency. Here, we describe a highly sensitive real-time PCR protocol that allows for the correct quantification of virtually all group-M HIV-1 strains with a higher degree of accuracy compared with other methods. The protocol involves three stages, namely DNA extraction/lysis, cellular DNA quantification and HIV-1 proviral load assessment. Owing to the robustness of the PCR design, this assay can be performed on crude cellular extracts, and therefore it may be suitable for the routine analysis of clinical samples even in developing countries. An accurate quantification of the HIV-1 proviral load can be achieved within 1 d from blood withdrawal.
Wu, Jinlong; Chen, Lin; Lin, Dingbo; Ma, Zhaocheng; Deng, Xiuxin
The effects of tissue type, harvest maturity, and genetic factors on the expression of genes that related to citrus fruit allergies remain poorly understood. In the present study, a multiplex real-time PCR assay was developed to monitor the expression of citrus allergen genes individually with the advantages of much fewer sample requirements and simultaneously multiple target genes detection. Gene specific primer pairs and Taqman probes of three citrus allergen genes Cit s 1.01, Cit s 2.01, and Cit s 3.01 and the house-keeping gene β-actin were designed based on gene sequence differences. The PCR results showed that differential expression patterns were found during the ripening process. The expression levels of Cit s 3.01 were much higher than those of Cit s 1.01 and Cit s 2.01 in both peel and pulp tissues among 10 citrus cultivars. Data suggested that Kao Phuang Pummelo could be safely consumed with a potential low risk in allergenicity. Considering that assessing allergenicity is one of the tests in food safety, this assay might also facilitate the breeding and production of "allergy-friendly" citrus fruits.
Fernández, Gema; Martró, Elisa; González, Victoria; Saludes, Verónica; Bascuñana, Elisabet; Marcó, Clara; Rivaya, Belén; López, Evelin; Coll, Pep; Matas, Lurdes; Ausina, Vicente
Sexually transmitted infections (STI) are currently on the increase worldwide. New molecular tools have been developed in the past few years in order to improve their diagnosis. An evaluation was carried out using a new commercially available real-time PCR assay, Anyplex™ II STI-7 (Seegene, Seoul, Korea), which detects seven major pathogens in a single reaction - Chlamydia trachomatis, Neisseria gonorrhoeae, Trichomonas vaginalis, Mycoplasma hominis, Mycoplasma genitalium, Ureaplasma urealyticum, and Ureaplasma parvum - and compared with conventional methods performed in our laboratory. Two different populations were included, and 267 specimens from different sites of infection (urines, endocervical swabs, rectal swabs, vaginal swabs, urethral swabs and one inguinal adenopathy) were processed for both methods. The parameters of clinical performance were calculated for C. trachomatis, N. gonorrhoeae, and T. vaginalis, and the assay achieved sensitivities (SE) from 93.94% to 100%, and specificities (SP) from 96.55% to 100%, with negative predictive values (NPV) from 93.33% to 98.85%, and positive predictive values (PPV) from 96.88% to 100%, with a very good agreement (kappa index from 0.88 to 1). Anyplex™ II STI-7 is a good tool for the reliable diagnosis of STI. Its ease of use and processing allows it to be incorporated into the day to day laboratory work. Copyright © 2015 Elsevier España, S.L.U. and Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica. All rights reserved.
Pereira, Mariana R.; Rocha-Silva, Fabiana; Graciele-Melo, Cidiane; Lafuente, Camila R.; Magalhães, Telcia; Caligiorne, Rachel B.
The diagnosis of visceral leishmaniasis (VL) is a challenging issue and several studies worldwide have evaluated the different tools to reach a diagnostic solution. The polymerase chain reaction (PCR) has proven to be effective in detecting the genome of Leishmania species in different biological samples. In this study, we compared the conventional PCR and real-time PCR using the Sybr Green system and their application in molecular diagnosis of visceral leishmaniasis in peripheral blood as a biological sample. The genus-specific conserved region of kinetoplast DNA (kDNA) was the target of amplification. We studied 30 samples from patients with suspect of visceral leishmaniasis who were treated by the Medical Clinic of Santa Casa de Belo Horizonte Hospital, Brazil. Among the samples studied, 19 had a confirmed diagnosis for VL by serology and/or by clinical findings. Among these 19 samples, 63% (n = 12) presented positive results for serology and 79% (n = 15) positive results in both PCR methodologies. This fact suggests that the PCR technique can assist in the diagnosis of visceral leishmaniasis in patients who do not have detectable antibodies by serology but can present the genome of the parasite circulating in whole blood. Also, it was possible to observe that there was conformity between the results of the techniques of cPCR and qPCR using the Sybr Green system in 100% of samples analyzed. These data suggest that both PCR techniques were equally effective for detection of the genome of the parasite in the patient's blood. PMID:24689047
Kitamura, Masanori; Hiramatsu, Nobuhiko
Endoplasmic reticulum (ER) stress is involved in a wide range of pathologies. Detection and monitoring of the unfolded protein response are required to disclose the link between ER stress and diseases. Assessment of ER stress is also essential for evaluation of therapeutic drugs in vitro and in vivo; that is, their therapeutic utility as well as adverse effects. For detection and monitoring of ER stress in living cells and animals, ER stress-responsive alkaline phosphatase (ESTRAP), also called secreted alkaline phosphatase (SEAP), serves as a useful indicator. In cells genetically engineered to express SEAP, secretion of SEAP is quickly downregulated in response to ER stress. This phenomenon is observed in a wide range of cell types triggered by various ER stress inducers. The magnitude of the decrease in extracellular SEAP is proportional to the intensity of ER stress, which is inversely correlated with the induction of endogenous ER stress markers. In contrast to SEAP, the activity of intracellular luciferase is not affected by ER stress. ER stress causes a decrease in SEAP activity not via transcriptional suppression but via abnormal posttranslational modification, accelerated degradation, and reduced secretion of SEAP protein. In mice constitutively producing SEAP, in vivo induction of ER stress similarly causes rapid reduction in serum SEAP activity. Using SEAP as an indicator, real-time monitoring of ER stress in living cells and animals is feasible. The ESTRAP method provides a powerful tool to investigate the pathogenesis of ER stress-associated diseases, to assess toxicity and the adverse effects of drugs, and to develop therapeutic agents for the treatment of ER stress-related disorders. Copyright © 2011 Elsevier Inc. All rights reserved.
van Coppenraet, E. S. Bruijnesteijn; Lindeboom, J. A.; Prins, J. M.; Peeters, M. F.; Claas, E. C. J.; Kuijper, E. J.
A real-time PCR assay was developed to diagnose and identify the causative agents of suspected mycobacterial lymphadenitis. Primers and probes for the real-time PCR were designed on the basis of the internal transcribed spacer sequence, enabling the recognition of the genus Mycobacterium and the species Mycobacterium avium and M. tuberculosis. The detection limit for the assay was established at 1,100 CFU/ml of pus, and the specificity tests showed no false-positive reaction with other mycobacterial species and other pathogens causing lymphadenitis. From 67 children with suspected mycobacterial lymphadenitis based on a positive mycobacterial skin test, 102 samples (58 fine-needle aspirates [FNA] and 44 tissue specimens) were obtained. The real-time PCR assay detected a mycobacterial infection in 48 patients (71.6%), whereas auramine staining and culturing were positive for 31 (46.3%) and 28 (41.8%) of the patients. The addition of the real-time PCR assay to conventional diagnostic tests resulted in the recognition of 13 more patients with mycobacterial disease. These results indicate that the real-time PCR is more sensitive than conventional staining and culturing techniques (P = 0.006). The M. avium-specific real-time PCR was positive for 38 patients, and the M. tuberculosis-specific real-time PCR was positive for 1 patient. Analysis of 27 patients from whom FNA and tissue biopsy specimens were collected revealed significantly more positive real-time PCR results for FNA than for tissue biopsy specimens (P = 0.003). Samples from an age-matched control group of 50 patients with PCR-proven cat scratch disease were all found to be negative by the real-time PCR. We conclude that this real-time PCR assay with a sensitivity of 72% for patients with lymphadenitis and a specificity of 100% for the detection of atypical mycobacteria can provide excellent support for clinical decision making in children with lymphadenitis. PMID:15184446
Koshy, Linda; Anju, A L; Harikrishnan, S; Kutty, V R; Jissa, V T; Kurikesu, Irin; Jayachandran, Parvathy; Jayakumaran Nair, A; Gangaprasad, A; Nair, G M; Sudhakaran, P R
The extraction of genomic DNA is the crucial first step in large-scale epidemiological studies. Though there are many popular DNA isolation methods from human whole blood, only a few reports have compared their efficiencies using both end-point and real-time PCR assays. Genomic DNA was extracted from coronary artery disease patients using solution-based conventional protocols such as the phenol-chloroform/proteinase-K method and a non-phenolic non-enzymatic Rapid-Method, which were evaluated and compared vis-a-vis a commercially available silica column-based Blood DNA isolation kit. The appropriate method for efficiently extracting relatively pure DNA was assessed based on the total DNA yield, concentration, purity ratios (A260/A280 and A260/A230), spectral profile and agarose gel electrophoresis analysis. The quality of the isolated DNA was further analysed for PCR inhibition using a murine specific ATP1A3 qPCR assay and mtDNA/Y-chromosome ratio determination assay. The suitability of the extracted DNA for downstream applications such as end-point SNP genotyping, was tested using PCR-RFLP analysis of the AGTR1-1166A>C variant, a mirSNP having pharmacogenetic relevance in cardiovascular diseases. Compared to the traditional phenol-chloroform/proteinase-K method, our results indicated the Rapid-Method to be a more suitable protocol for genomic DNA extraction from human whole blood in terms of DNA quantity, quality, safety, processing time and cost. The Rapid-Method, which is based on a simple salting-out procedure, is not only safe and cost-effective, but also has the added advantage of being scaled up to process variable sample volumes, thus enabling it to be applied in large-scale epidemiological studies.
Vester, Diana; Lagoda, Antje; Hoffmann, Diana; Seitz, Claudius; Heldt, Stefan; Bettenbrock, Katja; Genzel, Yvonne; Reichl, Udo
A quantitative real-time reverse transcriptase PCR (RT-qPCR) assay was developed for the analysis of influenza A virus transcription and replication dynamics in mammalian cell culture. The assay is based on a polarity- and sequence-specific reverse transcription used to distinguish specifically between viral genomes (vRNA(-)), replicative intermediates (cRNA(+)) and viral messenger RNAs (vmRNA(+)) of segments 4 (HA), 6 (NA), 7 (M) and 8 (NS) during the life cycle of influenza virus. Synthetic viral RNAs used as reference standards for validation and quantitation were prepared for each viral RNA type and segment. Assay validation demonstrated linearity over five orders of magnitude, sensitivity of 1.0 x 10(3) to 8.9 x 10(3) of viral RNA molecules, repeatability and reproducibility of less than 0.8-3.1% CV (coefficient of variation). Dynamics of influenza A virus infection in adherent MDCK cells, a substrate considered for human influenza vaccine manufacturing, were analyzed. In general, mainly vmRNA(+) were synthesized during early phases of infection at about 0.6 hpi, followed immediately by cRNA(+) synthesis and after a short delay of about 1.9 hpi viral genome replication could be detected. The vRNA(-)s were synthesized in equimolar amounts and similar dynamics whereas preferential synthesis of NS1 vmRNA(+) in early transcription phases and a delay for M1 vmRNA(+) was found. Copyright 2010 Elsevier B.V. All rights reserved.
Tocqueville, Véronique; Ferré, Séverine; Nguyen, Ngoc Hong Phuc; Kempf, Isabelle; Marois-Créhan, Corinne
A real-time TaqMan PCR assay based on the gene encoding the protein p37 was developed to detect Mycoplasma hyorhinis. Its specificity was validated with 29 epidemiologically unrelated M. hyorhinis strains (28 field strains and one reference strain) and other mycoplasma species or with other microorganisms commonly found in pigs. The estimated detection limit of this qPCR assay was 125 microorganism equivalents/μl. The same 29 epidemiologically unrelated M. hyorhinis strains and four previously fully sequenced strains were typed by two portable typing methods, the sequencing of the p37 gene and a multilocus sequence typing (MLST) scheme. The first method revealed 18 distinct nucleotide sequences and insufficient discriminatory power (0.934). The MLST scheme was developed with the sequenced genomes of the M. hyorhinis strains HUB-1, GDL-1, MCLD, and SK76 and based on the genes dnaA, rpoB, gyrB, gltX, adk, and gmk. In total, 2,304 bp of sequence was analyzed for each strain. MLST was capable of subdividing the 33 strains into 29 distinct sequence types. The discriminatory power of the method was >0.95, which is the threshold value for interpreting typing results with confidence (D=0.989). Population analysis showed that recombination in M. hyorhinis occurs and that strains are diverse but with a certain clonality (one unique clonal complex was identified). The new qPCR assay and the robust MLST scheme are available for the acquisition of new knowledge on M. hyorhinis epidemiology. A web-accessible database has been set up for the M. hyorhinis MLST scheme at http://pubmlst.org/mhyorhinis/.
Greeff, Mariska R; Christison, Kevin W; Macey, Brett M
Abalone Haliotis midae exhibiting typical clinical signs of tubercle mycosis were discovered in South African culture facilities in 2006, posing a significant threat to the industry. The fungus responsible for the outbreak was identified as a Peronosporomycete, Halioticida noduliformans. Currently, histopathology and gross observation are used to diagnose this disease, but these 2 methods are neither rapid nor sensitive enough to provide accurate and reliable diagnosis. Real-time quantitative PCR (qPCR) is a rapid and reliable method for the detection and quantification of a variety of pathogens, so therefore we aimed to develop a qPCR assay for species-specific detection and quantification of H. noduliformans. Effective extraction of H. noduliformans genomic DNA from laboratory grown cultures, as well as from spiked abalone tissues, was accomplished by grinding samples using a pellet pestle followed by heat lysis in the presence of Chelax-100 beads. A set of oligonucleotide primers was designed to specifically amplify H. noduliformans DNA in the large subunit (LSU) rRNA gene, and tested for cross-reactivity to DNA extracted from related and non-related fungi isolated from seaweeds, crustaceans and healthy abalone; no cross-amplification was detected. When performing PCR assays in an abalone tissue matrix, an environment designed to be a non-sterile simulation of environmental conditions, no amplification occurred in the negative controls. The qPCR assay sensitivity was determined to be approximately 0.28 pg of fungal DNA (~2.3 spores) in a 25 µl reaction volume. Our qPCR technique will be useful for monitoring and quantifying H. noduliformans for the surveillance and management of abalone tubercle mycosis in South Africa.
Hilbert, David W; Smith, William L; Chadwick, Sean G; Toner, Geoffrey; Mordechai, Eli; Adelson, Martin E; Aguin, Tina J; Sobel, Jack D; Gygax, Scott E
Bacterial vaginosis (BV) is the most common gynecological infection in the United States. Diagnosis based on Amsel's criteria can be challenging and can be aided by laboratory-based testing. A standard method for diagnosis in research studies is enumeration of bacterial morphotypes of a Gram-stained vaginal smear (i.e., Nugent scoring). However, this technique is subjective, requires specialized training, and is not widely available. Therefore, a highly accurate molecular assay for the diagnosis of BV would be of great utility. We analyzed 385 vaginal specimens collected prospectively from subjects who were evaluated for BV by clinical signs and Nugent scoring. We analyzed quantitative real-time PCR (qPCR) assays on DNA extracted from these specimens to quantify nine organisms associated with vaginal health or disease:Gardnerella vaginalis,Atopobium vaginae, BV-associated bacteria 2 (BVAB2, an uncultured member of the orderClostridiales),Megasphaeraphylotype 1 or 2,Lactobacillus iners,Lactobacillus crispatus,Lactobacillus gasseri, andLactobacillus jensenii We generated a logistic regression model that identifiedG. vaginalis,A. vaginae, andMegasphaeraphylotypes 1 and 2 as the organisms for which quantification provided the most accurate diagnosis of symptomatic BV, as defined by Amsel's criteria and Nugent scoring, with 92% sensitivity, 95% specificity, 94% positive predictive value, and 94% negative predictive value. The inclusion ofLactobacillusspp. did not contribute sufficiently to the quantitative model for symptomatic BV detection. This molecular assay is a highly accurate laboratory tool to assist in the diagnosis of symptomatic BV.
Sow, Doudou; Parola, Philippe; Sylla, Khadime; Ndiaye, Magatte; Delaunay, Pascal; Halfon, Philippe; Camiade, Sabine; Dieng, Thérèse; Tine, Roger C K; Faye, Babacar; Ndiaye, Jean Louis; Dieng, Yémou; Gaye, Oumar; Raoult, Didier; Bittar, Fadi
Gastrointestinal parasite infections represent one of the biggest public health problems in the world. Therefore, appropriate innovative tools are needed for assessing interventions to control these infections. This study aims to compare the performance of real-time polymerase chain reaction (PCR) assays to microscopic examination for detection of intestinal parasites. A direct microscopic examination and stool concentration was performed on 98 stool samples from patients attending Senegalese hospitals. Negative microscopic control samples were also collected in Nice and Marseille (France). Species-specific primers/probes were used to detect 20 common gastrointestinal protozoans and helminths. Positive frequency and the sensitivity of each real-time PCR assay were compared with conventional microscopic examination. Real-time PCR was positive in 72 of 98 samples (73.5%), whereas microscopic examination was positive in 37 (37.7%) samples (P < 0.001). The real-time PCR assays were more sensitive than microscopy, with 57.4% (31/54) versus 18.5% (10/54), respectively, in the detection of parasites in asymptomatic patients (P < 0.05). In terms of polyparasitism, there were more coinfections detected by real-time PCR assays compared with microscopic methods (25.5% versus 3.06%). In comparison to parasite prevalence on individual samples, the results showed a perfect agreement (100%) between the two techniques for seven species, whereas discrepancies were observed for the others (agreement percentage varying from 64.2% to 98.9%). Real-time PCR appeared to be superior to microscopic examination for the detection of parasites in stool samples. This assay will be useful in diagnostic laboratories and in the field for evaluating the efficacy of mass drug administration programs.
Development of Conventional and Real-Time Reverse Transcription Polymerase Chain Reaction Assays to Detect Tembusu Virus in Culex tarsalis Mosquitoes...culture supernatant and Culex tarsalismosquitoes. We used this assay to document the replication of TMUV in Cx. tarsalis, where titers increased 1,000...disease. Members of the genus Flavivirus (family Flaviviridae), including Japanese encephalitis virus (JEV), West Nile (WNV), yellow fever (YFV), and
Koepsell, Scott A.; Hinrichs, Steven H.
A real-time PCR assay to detect Histoplasma capsulatum in formalin-fixed, paraffin-embedded (FFPE) tissue is described. The assay had an analytical sensitivity of 6 pg/μl of fungal DNA, analytical specificity of 100%, and clinical sensitivity of 88.9%. This proof-of-concept study may aid in the diagnosis of histoplasmosis from FFPE tissue. PMID:22855519
Miller, Laura C; Crawford, Kimberly K; Lager, Kelly M; Kellner, Steven G; Brockmeier, Susan L
In April 2013, a Porcine epidemic diarrhea virus (PEDV) epidemic began in the United States. As part of the response, real-time reverse transcription polymerase chain reaction (RT-PCR) assays to detect PEDV were developed by several veterinary diagnostic laboratories. Our study evaluated RT-PCR PEDV assays that detect the N gene (gN) and S gene (gS) for their ability to detect PEDV infection and the transmission potential of pigs experimentally exposed to PEDV. Detection limits and quantification cycle (Cq) values of real-time RT-PCR were assayed for PEDV samples and positive controls for both gN and gS. The limit of detection for the gN assay was 10(-6) (mean Cq: 39.82 ± 0.30) and 10(-5) (mean Cq: 39.39 ± 0.72) for the gS assay with PEDV strain USA/Colorado/2013. Following recommended guidelines, rectal swabs (n = 1,064) were tested; 354 samples were positive by gN assay and 349 samples were positive by gS assay (Cq ≤ 34.99), 710 samples were negative by gN assay and 715 were negative by gS assay (Cq > 34.99) of which 355 and 344 were "undetermined" (i.e., undetected within a threshold of 40 RT-PCR cycles, by gN and gS assays, respectively). The coefficient of variation (intra-assay variation) ranged from 0.00% to 2.65% and interassay variation had an average of 2.75%. PEDV could be detected in rectal swabs from all pigs for ~2 weeks postinfection at which time the prevalence began to decrease until all pigs were RT-PCR negative by 5 weeks postinfection. Our study demonstrated that RT-PCR assays functioned well to detect PEDV and that the gN assay was slightly better.
Birdsell, Dawn N.; Vogler, Amy J.; Buchhagen, Jordan; Clare, Ashley; Kaufman, Emily; Naumann, Amber; Driebe, Elizabeth; Wagner, David M.; Keim, Paul S.
Francisella tularensis, the etiologic agent of tularemia and a Class A Select Agent, is divided into three subspecies and multiple subpopulations that differ in virulence and geographic distribution. Given these differences, there is a need to rapidly and accurately determine if a strain is F. tularensis and, if it is, assign it to subspecies and subpopulation. We designed TaqMan real-time PCR genotyping assays using eleven single nucleotide polymorphisms (SNPs) that were potentially specific to closely related groups within the genus Francisella, including numerous subpopulations within F. tularensis species. We performed extensive validation studies to test the specificity of these SNPs to particular populations by screening the assays across a set of 565 genetically and geographically diverse F. tularensis isolates and an additional 21 genetic near-neighbor (outgroup) isolates. All eleven assays correctly determined the genetic groups of all 565 F. tularensis isolates. One assay differentiates F. tularensis, F. novicida, and F. hispaniensis from the more genetically distant F. philomiragia and Francisella-like endosymbionts. Another assay differentiates F. tularensis isolates from near neighbors. The remaining nine assays classify F. tularensis-confirmed isolates into F. tularensis subspecies and subpopulations. The genotyping accuracy of these nine assays diminished when tested on outgroup isolates (i.e. non F. tularensis), therefore a hierarchical approach of assay usage is recommended wherein the F. tularensis-specific assay is used before the nine downstream assays. Among F. tularensis isolates, all eleven assays were highly sensitive, consistently amplifying very low concentrations of DNA. Altogether, these eleven TaqMan real-time PCR assays represent a highly accurate, rapid, and sensitive means of identifying the species, subspecies, and subpopulation of any F. tularensis isolate if used in a step-wise hierarchical scheme. These assays would be very
Vidya, Madhavan; Saravanan, Shanmugam; Rifkin, Samara; Solomon, Sunil S; Waldrop, Greer; Mayer, Kenneth H; Solomon, Suniti; Balakrishnan, Pachamuthu
High costs and stringent requirements for storage and transport of plasma, often prohibit the availability of HIV viral load quantification in resource-limited settings. Dried blood spots (DBS) represent a better method of specimen collection that removes many of these logistical and technical limitations. The present study aimed to assess the performance of the Abbott m2000rt assay for quantitation of HIV-1 RNA in DBS specimens using plasma as a "gold standard" for comparison. One hundred paired DBS and plasma specimens were collected from patients infected with HIV, who were 18 years and older during routine visits to a private tertiary-care clinic in Chennai, India. HIV-1 RNA was extracted manually and then detected using the m2000rt assay. The mean plasma and DBS viral loads were 4.27 (95% CI: 2.65, 5.88) and 4.14 (95% CI: 1.96, 6.32) log copies/mL, respectively. The overall sensitivity of DBS reached 95%; with sensitivities of 62%, 88% and 100% when stratified by viral load ranges of ≤1000, 1000-3000 and >3000 copies/mL, respectively. An over quantitation of the viral load with DBS was observed in pairs with plasma viral load<3000 copies/mL [d=-0.3 log copies/mL (ranging from -0.1 to 0.6 log copies/mL)]. The study showed a strong concordance in RNA levels between plasma and DBS. The use of DBS specimens should be considered for HIV monitoring and for detection of virologic failure in resource-limited settings. Copyright © 2012 Elsevier B.V. All rights reserved.
Zemtsova, Galina E; Montgomery, Merrill; Levin, Michael L
Studies on the natural transmission cycles of zoonotic pathogens and the reservoir competence of vertebrate hosts require methods for reliable diagnosis of infection in wild and laboratory animals. Several PCR-based applications have been developed for detection of infections caused by Spotted Fever group Rickettsia spp. in a variety of animal tissues. These assays are being widely used by researchers, but they differ in their sensitivity and reliability. We compared the sensitivity of five previously published conventional PCR assays and one SYBR green-based real-time PCR assay for the detection of rickettsial DNA in blood and tissue samples from Rickettsia- infected laboratory animals (n = 87). The real-time PCR, which detected rickettsial DNA in 37.9% of samples, was the most sensitive. The next best were the semi-nested ompA assay and rpoB conventional PCR, which detected as positive 18.4% and 14.9% samples respectively. Conventional assays targeting ompB, gltA and hrtA genes have been the least sensitive. Therefore, we recommend the SYBR green-based real-time PCR as a tool for the detection of rickettsial DNA in animal samples due to its higher sensitivity when compared to more traditional assays.
Bohaychuk, V M; Gensler, G E; McFall, M E; King, R K; Renter, D G
Conventional culture methods have traditionally been considered the "gold standards" for the isolation and identification of foodborne pathogens. However, culture methods are labor-intensive and time-consuming. We have developed a real-time PCR assay for the detection of Salmonella in a variety of food and food-animal matrices. The real-time PCR assay incorporates both primers and hybridization probes based on the sequence of the Salmonella invA gene and uses fluorescent resonance energy transfer technology to ensure highly sensitive and specific results. This method correctly classified 51 laboratory isolates of Salmonella and 28 non-Salmonella strains. The method was also validated with a large number of field samples that consisted of porcine feces and cecal contents, pork carcasses, bovine feces and beef carcasses, poultry cecal contents and carcasses, equine feces, animal feeds, and various food products. The samples (3388) were preenriched in buffered peptone water and then selectively enriched in tetrathionate and Rappaport-Vassiliadis broths. Aliquots of the selective enrichment broths were combined for DNA extraction and analysis by the real-time PCR assay. When compared with the culture method, the diagnostic sensitivity of the PCR assay for the various matrices ranged from 97.1 to 100.0%, and the diagnostic specificity ranged from 91.3 to 100.0%. Kappa values ranged from 0.87 to 1.00, indicating excellent agreement of the real-time PCR assay to the culture method. The reduction in time and labor makes this highly sensitive and specific real-time PCR assay an excellent alternative to conventional culture methods for surveillance and research studies to improve food safety.
Brito, Barbara P; Gardner, Ian A; Hietala, Sharon K; Crossley, Beate M
Bluetongue is a vector-borne viral disease that affects domestic and wild ruminants. The epidemiology of this disease has recently changed, with occurrence in new geographic areas. Various real-time quantitative reverse transcription polymerase chain reaction (real-time qRT-PCR) assays are used to detect Bluetongue virus (BTV); however, the impact of biologic differences between New World camelids and domestic ruminant samples on PCR efficiency, for which the BTV real-time qRT-PCR was initially validated are unknown. New world camelids are known to have important biologic differences in whole blood composition, including hemoglobin concentration, which can alter PCR performance. In the present study, sheep, cattle, and alpaca blood were spiked with BTV serotypes 10, 11, 13, and 17 and analyzed in 10-fold dilutions by real-time qRT-PCR to determine if species affected nucleic acid recovery and assay performance. A separate experiment was performed using spiked alpaca blood subsequently diluted in 10-fold series in sheep blood to assess the influence of alpaca blood on performance efficiency of the BTV real-time qRT-PCR assay. Results showed that BTV-specific nucleic acid detection from alpaca blood was consistently 1-2 logs lower than from sheep and cattle blood, and results were similar for each of the 4 BTV serotypes analyzed.
Hodgetts, Jennifer; Boonham, Neil; Mumford, Rick; Dickinson, Matthew
Primers and probes based on the 23S rRNA gene have been utilized to design a range of real-time PCR assays for routine phytoplasma diagnostics. These assays have been authenticated as phytoplasma specific and shown to be at least as sensitive as nested PCR. A universal assay to detect all phytoplasmas has been developed, along with a multiplex assay to discriminate 16SrI group phytoplasmas from members of all of the other 16Sr groups. Assays for the 16SrII, 16SrIV, and 16SrXII groups have also been developed to confirm that the 23S rRNA gene can be used to design group-specific assays. PMID:19270148
Thurman, Kathleen A; Warner, Agnes K; Cowart, Kelley C; Benitez, Alvaro J; Winchell, Jonas M
A multiplex real-time PCR assay for the detection of Mycoplasma pneumoniae (MP181), Chlamydia (Chlamydophila) pneumoniae (CP-Arg), Legionella spp. (Pan-Leg), and the human RNase P (RNase P) gene was developed for rapid testing of atypical bacterial respiratory pathogens in clinical specimens. This method uses 4 distinct hydrolysis probes to detect 3 leading causes of community-acquired pneumonia. The assay was evaluated for specificity and sensitivity by testing against 35 related organisms, a dilution series of each specific target and 197 clinical specimens. Specificity testing demonstrated no cross-reactivity. A comparison to previously validated singleplex real-time PCR assays for each agent was also performed. The analytical sensitivity for specific pathogen targets in both the singleplex and multiplex was identical (50 fg), while efficiencies ranged from 82% to 97% for the singleplex assays and from 90% to 100% for the multiplex assay. The clinical sensitivity of the multiplex assay was improved for the Pan-Leg and CP-Arg targets when compared to the singleplex. The MP181 assay displayed equivalent performance. This multiplex assay provides an overall improvement in the diagnostic capability for these agents by demonstrating a sensitive, high-throughput and rapid method. This procedure may allow for a practical and efficient means to test respiratory clinical specimens for atypical pneumonia agents in health care settings and facilitate an appropriate public health response to outbreaks.
Vera-Jimenez, N I; Pietretti, D; Wiegertjes, G F; Nielsen, M E
The respiratory burst is an important feature of the immune system. The increase in cellular oxygen uptake that marks the initiation of the respiratory burst is followed by the production of reactive oxygen species (ROS) such as superoxide anion and hydrogen peroxide which plays a role in the clearance of pathogens and tissue regeneration processes. Therefore, the respiratory burst and associated ROS constitute important indicators of fish health status. This paper compares two methods for quantitation of ROS produced during the respiratory burst in common carp: the widely used, single-point measurement based on the intracellular reduction of nitroblue tetrazolium (NBT) and a real-time luminol-enhanced assay based on the detection of native chemiluminescence. Both assays allowed for detection of dose-dependent changes in magnitude of the respiratory burst response induced by β-glucans in head kidney cells of carp. However, whereas the NBT assay was shown to detect the production of only superoxide anions, the real-time luminol-enhanced assay could detect the production of both superoxide anions and hydrogen peroxide. Only the chemiluminescence assay could reliably record the production of ROS on a real-time scale at frequent and continual time intervals for time course experiments, providing more detailed information on the respiratory burst response. The real-time chemiluminescence assay was used to measure respiratory burst activity in macrophage and neutrophilic granulocyte-enriched head kidney cell fractions and total head kidney cell suspensions and proved to be a fast, reliable, automated multiwell microplate assay to quantitate fish health status modulated by β-glucans.
Park, Ki-Hyun; Park, Hyesun; Kim, Myungshin; Kim, Yonggoo; Han, Kyungja; Oh, Eun-Jee
Although real-time cell electronic sensing (RT-CES) system-based natural killer (NK) cytotoxicity has been introduced, it has not been evaluated using human blood samples. In present study, we measured flowcytometry based assay (FCA) and RT-CES based NK cytotoxicity and analyzed degranulation activity (CD107a) and cytokine production. In 98 healthy individuals, FCA with peripheral blood mononuclear cells (PBMCs) at effector to target (E/T) ratio of 32 revealed 46.5 ± 2.6% cytolysis of K562 cells, and 23.5 ± 1.1% of NK cells showed increased degranulation. In RT-CES system, adherent NIH3T3 target cells were resistant to basal killing by PBMC or NK cells. NK cell activation by adding IL-2 demonstrated real-time dynamic killing activity, and lymphokine-activated PBMC (E/T ratio of 32) from 15 individuals showed 59.1 ± 6.2% cytotoxicity results after 4 hours incubation in RT-CES system. However, there was no significant correlation between FCA and RT-CES cytotoxicity. After K562 target cell stimulation, PBMC produced profound proinflammatory and immunoregulatory cytokines/chemokines including IL-2, IL-8, IL-10, MIP-1α β, IFN-γ, and TNF-α, and cytokine/chemokine secretion was related to flowcytometry-based NK cytotoxicity. These data suggest that RT-CES and FCA differ in sensitivity, applicability and providing information, and further investigations are necessary in variable clinical conditions. PMID:24236291
Applewhite, Erain; Hirsch, Lesley
The New Jersey Supreme Court's 1998 ruling in Abbott v. Burke represents the first judicial directive in the nation that public education must include a high-quality, well-planned preschool program starting at age three. This decision applies to 30 urban school districts, known as the Abbott districts, that serve approximately 25 percent of the…
... Cardiovascular Systems, Inc., (Cardiovascular Devices), Riverside County, CA Pursuant to its Authority Under the... 153, has requested manufacturing authority on behalf of Abbott Cardiovascular Systems, Inc., within... procedures at sites within FTZ 153, on behalf of Abbott Cardiovascular Systems, Inc., as described in the...
Barnett, W. Steven; Jung, Kwanghee; Youn, Min-Jong; Frede, Ellen C.
New Jersey's Abbott Preschool program is of broad national and international interest because the Abbott program provides a model for building a high-quality system of universal pre-K through public-private partnerships that transform the existing system. The program offers high-quality pre-K to all children in 31 New Jersey communities with high…
... Foreign-Trade Zones Board Foreign-Trade Zone 22--Chicago, IL, Notification of Proposed Production Activity, Abbott Laboratories, Inc., AbbVie, Inc. (Pharmaceutical Production), North Chicago, IL, Area Abbott... authority within Subzones 22F and 22S, at sites located in the North Chicago and Lake County, Illinois, area...
Barnett, W. Steven; Jung, Kwanghee; Youn, Min-Jong; Frede, Ellen C.
New Jersey's Abbott Preschool program is of broad national and international interest because the Abbott program provides a model for building a high-quality system of universal pre-K through public-private partnerships that transform the existing system. The program offers high-quality pre-K to all children in 31 New Jersey communities with high…
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
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.
Porcine parvovirus 4 (PPV4) is a DNA virus, and a member of the Parvoviridae family within the Bocavirus genera. It was recently detected in swine, but its epidemiology and pathology remain unclear. A TaqMan-based real-time polymerase chain reaction (qPCR) assay targeting a conserved region of the O...
A multiplex Taqman®-based real-time reverse transcription (RT) polymerase chain reaction (PCR) assay was developed to detect all strains of Citrus tristeza virus (CTV) and to identify potentially severe strains of the virus. A CTV TaqMan probe (CTV-CY5) based on the coat protein (CP) gene sequences...
Kim, Miju; Yoo, Insuk; Lee, Shin-Young; Hong, Yeun; Kim, Hae-Yeong
The TaqMan® real-time PCR assay using the mitochondrial D-loop region was developed for the quantitative detection of pork in processed meat products. The newly designed primers and probe specifically amplified pork without any cross-reactivity with non-target animal species. The limit of detection of the real-time PCR assay was 0.1pg of heat-treated pork meat and 0.1% (w/w) pork meat in beef and chicken meat mixtures. The quantitative real-time PCR assay was applied to analyze the pork meat content in 22 commercial processed meat products including jerkies, press hams, sausages, hamburger patties and steaks, grilled short rib patties, and nuggets. The developed real-time PCR method was able to detect pork meat in various types of processed meat products that declared the use of pork meat on their label. All processed meat products that declared no use of pork meat showed a negative result in the assay. The method developed in this study showed sensitivity and specificity in the quantification of pork meat in commercial processed meat products.
Palle-Reisch, Monika; Cichna-Markl, Margit; Hochegger, Rupert
The paper presents a duplex real-time PCR assay for the simultaneous detection of three potentially allergenic mustard species commonly used in food: white mustard (Sinapis alba), black mustard (Brassica nigra) and brown mustard (Brassica juncea). White mustard is detected in the "green" and black/brown mustard in the "yellow" channel. The duplex real-time PCR assay does not show cross-reactivity with other Brassicaceae species including broccoli, cauliflower, radish and rapeseed. Low cross-reactivities (difference in the Ct value ⩾ 11.91 compared with the positive control) were obtained with cumin, fenugreek, ginger, rye and turmeric. When applying 500 ng DNA per PCR tube, the duplex real-time PCR assay allowed the detection of white, black and brown mustard in brewed model sausages down to a concentration of 5mg/kg in 10 out of 10 replicates. The duplex real-time PCR assay was applied to verify correct labelling of commercial foodstuffs. Copyright © 2013 Elsevier Ltd. All rights reserved.
A quantitative real-time polymerase chain reaction (PCR) assay for sulfate-reducing bacteria (SRB) was developed that targeted the dissimilatory sulfite reductase gene (dsrA). Degenerate primer sets were developed to detect three different groups of SRB in stored swine manure using a SYBR Green qua...
Researchers have proposed the adoption of 3 distinct genetic taxa among bacteria previously classified as Edwardsiella tarda; namely E. tarda, E. piscicida, and a taxon presently termed E. piscicida–like. Individual real-time polymerase chain reaction (qPCR) assays were developed, based on published...
Decousser, Jean-Winoc; Methlouthi, Imen; Pina, Patrick; Collignon, Anne; Allouch, Pierre
A real-time PCR assay with locked nucleic acid probes was developed to screen mutations at codons 79 and 83 of the Streptococcus pneumoniae parC gene. Only silent mutations were detected among 236 French invasive fluoroquinolone-susceptible strains. This test could be useful for some high-risk patients or in national surveys. PMID:16569894
Zhao, Yanan; Petraitiene, Ruta; Walsh, Thomas J; Perlin, David S
A species-specific molecular beacon real-time PCR assay was developed for rapid diagnosis of Exserohilum rostratum infection. As low as 100 fg of E. rostratum DNA can be reliably detected in the presence of 50 ng of human DNA, with a dynamic linear quantification range from 20 ng to 200 fg.
Molecular detection methods such as PCR have been extensively used to type Cryptosporidium oocysts detected in the environment. More recently, studies have developed quantitative real-time PCR assays for detection and quantification of microbial contaminants in water as well as ...
Molecular detection methods such as PCR have been extensively used to type Cryptosporidium oocysts detected in the environment. More recently, studies have developed quantitative real-time PCR assays for detection and quantification of microbial contaminants in water as well as ...
Researchers have proposed the adoption of 3 distinct genetic taxa among bacteria previously classified as Edwardsiella tarda; namely E. tarda, E. piscicida, and a taxon presently termed E. piscicida–like. Individual real-time polymerase chain reaction (qPCR) assays were developed, based on published...
Researchers have proposed the adoption of 3 distinct genetic taxa among bacteria previously classified as Edwardsiella tarda; namely E. tarda, E. piscicida, and a taxon presently termed E. piscicida–like. Individual real-time polymerase chain reaction (qPCR) assays were developed, based on previousl...
the new assays. Type-specific TaqMan real-time PCR assays were designed and used independently to successfully identify 16 representative specimens...react with other adenoviruses outside of species HAdV-C, respiratory syncytial virus, influenza , or respiratory disease causing bacteria. These assays...rapid individual and public health responses. Results LightCycler and J.B.A.I.D.S. TaqMan real-time PCR assays The detection of Human adenoviruses C1
Ben Shabat, M; Meir, R; Haddas, R; Lapin, E; Shkoda, I; Raibstein, I; Perk, S; Davidson, I
Avian influenza viruses (AIVs) of the H9N2 subtype are a major economic problem in the poultry industry in Israel. Most field isolates from the last decade differ significantly from H9N2 isolates from Europe and the USA, rendering published detection methods inadequate. This study aimed to develop a real-time TaqMan((R)) RT-PCR assay, based on a conserved region in the HA9 gene. The assay was validated with viruses representing different genetic subtypes and other common avian pathogens, and was found specific to H9N2. The real-time RT-PCR assay was compared to RT-PCR, which is in routine diagnostic use. Real-time RT-PCR was found to be more sensitive than RT-PCR by 1.5-2.5 orders of magnitude when testing tracheal swabs directly and by 2-3 orders of magnitude allantoic fluid after AIV propagation in embryonated eggs. Sensitivity was quantified by using 10-fold dilutions of the H9-gene amplification fragment, and real-time RT-PCR was found to be 10(4)-fold more sensitive than RT-PCR. Clinical samples, which included tracheal and cloacal swabs, as well as allantoic fluid, were tested by both methods. By real-time RT-PCR 20% more positive H9N2 samples were detected than by RT-PCR. The real-time RT-PCR assay was found suitable for detection and epidemiological survey not only of Israeli H9N2 viruses, but also for isolates from other parts of the world.
Klein, Eileen J.; Galanakis, Emmanouil; Thomas, Anita A.; Stapp, Jennifer R.; Rich, Shannon; Buccat, Anne Marie; Tarr, Phillip I.
Timely accurate diagnosis of Shiga toxin-producing Escherichia coli (STEC) infections is important. We evaluated a laboratory-developed real-time PCR (LD-PCR) assay targeting stx1, stx2, and rfbEO157 with 2,386 qualifying stool samples submitted to the microbiology laboratory of a tertiary care pediatric center between July 2011 and December 2013. Broth cultures of PCR-positive samples were tested for Shiga toxins by enzyme immunoassay (EIA) (ImmunoCard STAT! enterohemorrhagic E. coli [EHEC]; Meridian Bioscience) and cultured in attempts to recover both O157 and non-O157 STEC. E. coli O157 and non-O157 STEC were detected in 35 and 18 cases, respectively. Hemolytic uremic syndrome (HUS) occurred in 12 patients (10 infected with STEC O157, one infected with STEC O125ac, and one with PCR evidence of STEC but no resulting isolate). Among the 59 PCR-positive STEC specimens from 53 patients, only 29 (54.7%) of the associated specimens were toxin positive by EIA. LD-PCR differentiated STEC O157 from non-O157 using rfbEO157, and LD-PCR results prompted successful recovery of E. coli O157 (n = 25) and non-O157 STEC (n = 8) isolates, although the primary cultures and toxin assays were frequently negative. A rapid “mega”-multiplex PCR (FilmArray gastrointestinal panel; BioFire Diagnostics) was used retrospectively, and results correlated with LD-PCR findings in 25 (89%) of the 28 sorbitol-MacConkey agar culture-negative STEC cases. These findings demonstrate that PCR is more sensitive than EIA and/or culture and distinguishes between O157 and non-O157 STEC in clinical samples and that E. coli O157:H7 remains the predominant cause of HUS in our institution. PCR is highly recommended for rapid diagnosis of pediatric STEC infections. PMID:25926491
Donà, Valentina; Kasraian, Sara; Lupo, Agnese; Guilarte, Yuvia N.; Hauser, Christoph; Furrer, Hansjakob; Unemo, Magnus; Low, Nicola
Resistance to antibiotics used against Neisseria gonorrhoeae infections is a major public health concern. Antimicrobial resistance (AMR) testing relies on time-consuming culture-based methods. Development of rapid molecular tests for detection of AMR determinants could provide valuable tools for surveillance and epidemiological studies and for informing individual case management. We developed a fast (<1.5-h) SYBR green-based real-time PCR method with high-resolution melting (HRM) analysis. One triplex and three duplex reactions included two sequences for N. gonorrhoeae identification and seven determinants of resistance to extended-spectrum cephalosporins (ESCs), azithromycin, ciprofloxacin, and spectinomycin. The method was validated by testing 39 previously fully characterized N. gonorrhoeae strains, 19 commensal Neisseria species strains, and an additional panel of 193 gonococcal isolates. Results were compared with results of culture-based AMR determination. The assay correctly identified N. gonorrhoeae and the presence or absence of the seven AMR determinants. There was some cross-reactivity with nongonococcal Neisseria species, and the detection limit was 103 to 104 genomic DNA (gDNA) copies/reaction. Overall, the platform accurately detected resistance to ciprofloxacin (sensitivity and specificity, 100%), ceftriaxone (sensitivity, 100%; specificity, 90%), cefixime (sensitivity, 92%; specificity, 94%), azithromycin (sensitivity and specificity, 100%), and spectinomycin (sensitivity and specificity, 100%). In conclusion, our methodology accurately detects mutations that generate resistance to antibiotics used to treat gonorrhea. Low assay sensitivity prevents direct diagnostic testing of clinical specimens, but this method can be used to screen collections of gonococcal isolates for AMR more quickly than current culture-based AMR testing. PMID:27225407
Donà, Valentina; Kasraian, Sara; Lupo, Agnese; Guilarte, Yuvia N; Hauser, Christoph; Furrer, Hansjakob; Unemo, Magnus; Low, Nicola; Endimiani, Andrea
Resistance to antibiotics used against Neisseria gonorrhoeae infections is a major public health concern. Antimicrobial resistance (AMR) testing relies on time-consuming culture-based methods. Development of rapid molecular tests for detection of AMR determinants could provide valuable tools for surveillance and epidemiological studies and for informing individual case management. We developed a fast (<1.5-h) SYBR green-based real-time PCR method with high-resolution melting (HRM) analysis. One triplex and three duplex reactions included two sequences for N. gonorrhoeae identification and seven determinants of resistance to extended-spectrum cephalosporins (ESCs), azithromycin, ciprofloxacin, and spectinomycin. The method was validated by testing 39 previously fully characterized N. gonorrhoeae strains, 19 commensal Neisseria species strains, and an additional panel of 193 gonococcal isolates. Results were compared with results of culture-based AMR determination. The assay correctly identified N. gonorrhoeae and the presence or absence of the seven AMR determinants. There was some cross-reactivity with nongonococcal Neisseria species, and the detection limit was 10(3) to 10(4) genomic DNA (gDNA) copies/reaction. Overall, the platform accurately detected resistance to ciprofloxacin (sensitivity and specificity, 100%), ceftriaxone (sensitivity, 100%; specificity, 90%), cefixime (sensitivity, 92%; specificity, 94%), azithromycin (sensitivity and specificity, 100%), and spectinomycin (sensitivity and specificity, 100%). In conclusion, our methodology accurately detects mutations that generate resistance to antibiotics used to treat gonorrhea. Low assay sensitivity prevents direct diagnostic testing of clinical specimens, but this method can be used to screen collections of gonococcal isolates for AMR more quickly than current culture-based AMR testing.
Akbulut, D.; Grant, K. A.; McLauchlin, J.
An upsurge in wound infections due to Clostridium botulinum and Clostridium tetani among users of illegal injected drugs (IDUs) occurred in the United Kingdom during 2003 and 2004. A real-time PCR assay was developed to detect a fragment of the neurotoxin gene of C. tetani (TeNT) and was used in conjunction with previously described assays for C. botulinum neurotoxin types A, B, and E (BoNTA, -B, and -E). The assays were sensitive, specific, rapid to perform, and applicable to investigating infections among IDUs using DNA extracted directly from wound tissue, as well as bacteria growing among mixed microflora in enrichment cultures and in pure culture on solid media. A combination of bioassay and PCR test results confirmed the clinical diagnosis in 10 of 25 cases of suspected botulism and two of five suspected cases of tetanus among IDUs. The PCR assays were in almost complete agreement with the conventional bioassays when considering results from different samples collected from the same patient. The replacement of bioassays by real-time PCR for the isolation and identification of both C. botulinum and C. tetani demonstrates a sensitivity and specificity similar to those of conventional approaches. However, the real-time PCR assays substantially improves the diagnostic process in terms of the speed of results and by the replacement of experimental animals. Recommendations are given for an improved strategy for the laboratory investigation of suspected wound botulism and tetanus among IDUs. PMID:16145075
Akbulut, D; Grant, K A; McLauchlin, J
An upsurge in wound infections due to Clostridium botulinum and Clostridium tetani among users of illegal injected drugs (IDUs) occurred in the United Kingdom during 2003 and 2004. A real-time PCR assay was developed to detect a fragment of the neurotoxin gene of C. tetani (TeNT) and was used in conjunction with previously described assays for C. botulinum neurotoxin types A, B, and E (BoNTA, -B, and -E). The assays were sensitive, specific, rapid to perform, and applicable to investigating infections among IDUs using DNA extracted directly from wound tissue, as well as bacteria growing among mixed microflora in enrichment cultures and in pure culture on solid media. A combination of bioassay and PCR test results confirmed the clinical diagnosis in 10 of 25 cases of suspected botulism and two of five suspected cases of tetanus among IDUs. The PCR assays were in almost complete agreement with the conventional bioassays when considering results from different samples collected from the same patient. The replacement of bioassays by real-time PCR for the isolation and identification of both C. botulinum and C. tetani demonstrates a sensitivity and specificity similar to those of conventional approaches. However, the real-time PCR assays substantially improves the diagnostic process in terms of the speed of results and by the replacement of experimental animals. Recommendations are given for an improved strategy for the laboratory investigation of suspected wound botulism and tetanus among IDUs.
Nordgren, Johan; Bucardo, Filemón; Dienus, Olaf; Svensson, Lennart; Lindgren, Per-Eric
Norovirus is now recognized as the leading cause of nonbacterial acute gastroenteritis in adults, causing numerous outbreaks worldwide. We have developed two novel light-upon-extension (LUX) real-time PCR assays for detection and quantification of norovirus genogroups I and II. The LUX system uses a fluorophore attached to one primer having a self-quenching hairpin structure, making it cost-effective and specific. The assays were evaluated against clinical stool specimens (n = 103) from Sweden and Nicaragua and compared to established methods. The norovirus assay detected more positive stool specimens (47/103) than conventional PCR (39/103) and corresponded to a TaqMan real-time PCR, with the exception of one specimen. Furthermore, the assays correctly identified all (n = 11) coded control specimens in a reference panel containing various genogroups and genotypes. Both LUX real-time PCR assays had a wide dynamic range, detecting from ≤101 to 107 genes per reaction, resulting in a theoretical lower limit of ≤∼20 000 viruses per gram of stool. No cross-reactivity was noticed with specimens containing other enteric viruses, and by using melting curve analysis we could differentiate between norovirus genogroups I and II. PMID:17959761
Jamnikar Ciglenečki, Urška; Toplak, Ivan
Real-time polymerase chain reaction (real-time PCR) is an accurate, rapid and reliable method that can be used for the detection and also for the quantitation of specific DNA molecules. It can be non-specific, with intercalating dyes (SYBR Green I dye) able to bind to any dsDNA, or specific with a probe (TaqMan), whereby the probe is designed to bind within the amplified PCR fragment. A new real-time reverse transcription and polymerase chain reaction (real time RT-PCR) assay with TaqMan probe for specific detection of acute bee paralysis virus was designed. The assay was optimized to be highly sensitive and analytically specific and tested with a selection of genetically diverse ABPV strains originating from Slovenia, the United Kingdom (UK), Hungary and Germany. The detection limit of the assay and sensitivity comparisons with conventional RT-PCR were analyzed and this assay can detect a minimum of 44 copies of ABPV/reaction and is 230 times more sensitive than conventional RT-PCR. In addition, the assay is highly reproducible, with an average slope of standard curve made of ten-fold dilutions of standard copies/reaction -3.479±0.19 and an average slope of standard curve made of ten-fold dilutions of RNA -3.409±0.18.
Balne, P K; Basu, S; Rath, S; Barik, M R; Sharma, S
This study is a comparative evaluation (Chi-square test) of a closed tube loop mediated isothermal amplification assay using hydroxy naphthol blue dye (HNB-LAMP), real-time polymerase chain reaction (PCR) and conventional PCR in the diagnosis of intraocular tuberculosis. Considering clinical presentation as the gold standard in 33 patients, the sensitivity of HNB-LAMP assay (75.8%) was higher (not significant, P value 0.2) than conventional PCR (57.6%) and lower than real-time PCR (90.9%). Specificity was 100% by all three methods. No amplification was observed in negative controls (n = 20) by all three methods. The cost of the HNB-LAMP assay was Rs. 500.00 and it does not require thermocycler, therefore, it can be used as an alternative to conventional PCR in resource-poor settings.
Dalpke, Alexander H; Hofko, Marjeta; Zimmermann, Stefan
Pneumocystis jirovecii is an opportunistic pathogen in immunocompromised and AIDS patients. Detection by quantitative PCR is faster and more sensitive than microscopic diagnosis yet requires specific infrastructure. We adapted a real-time PCR amplifying the major surface glycoprotein (MSG) target from Pneumocystis jirovecii for use on the new BD MAX platform. The assay allowed fully automated DNA extraction and multiplex real-time PCR. The BD MAX assay was evaluated against manual DNA extraction and conventional real-time PCR. The BD MAX was used in the research mode running a multiplex PCR (MSG, internal control, and sample process control). The assay had a detection limit of 10 copies of an MSG-encoding plasmid per PCR that equated to 500 copies/ml in respiratory specimens. We observed accurate quantification of MSG targets over a 7- to 8-log range. Prealiquoting and sealing of the complete PCR reagents in conical tubes allowed easy and convenient handling of the BD MAX PCR. In a retrospective analysis of 54 positive samples, the BD MAX assay showed good quantitative correlation with the reference PCR method (R(2) = 0.82). Cross-contamination was not observed. Prospectively, 278 respiratory samples were analyzed by both molecular assays. The positivity rate overall was 18.3%. The BD MAX assay identified 46 positive samples, compared to 40 by the reference PCR. The BD MAX assay required liquefaction of highly viscous samples with dithiothreitol as the only manual step, thus offering advantages for timely availability of molecular-based detection assays.
Hofko, Marjeta; Zimmermann, Stefan
Pneumocystis jirovecii is an opportunistic pathogen in immunocompromised and AIDS patients. Detection by quantitative PCR is faster and more sensitive than microscopic diagnosis yet requires specific infrastructure. We adapted a real-time PCR amplifying the major surface glycoprotein (MSG) target from Pneumocystis jirovecii for use on the new BD MAX platform. The assay allowed fully automated DNA extraction and multiplex real-time PCR. The BD MAX assay was evaluated against manual DNA extraction and conventional real-time PCR. The BD MAX was used in the research mode running a multiplex PCR (MSG, internal control, and sample process control). The assay had a detection limit of 10 copies of an MSG-encoding plasmid per PCR that equated to 500 copies/ml in respiratory specimens. We observed accurate quantification of MSG targets over a 7- to 8-log range. Prealiquoting and sealing of the complete PCR reagents in conical tubes allowed easy and convenient handling of the BD MAX PCR. In a retrospective analysis of 54 positive samples, the BD MAX assay showed good quantitative correlation with the reference PCR method (R2 = 0.82). Cross-contamination was not observed. Prospectively, 278 respiratory samples were analyzed by both molecular assays. The positivity rate overall was 18.3%. The BD MAX assay identified 46 positive samples, compared to 40 by the reference PCR. The BD MAX assay required liquefaction of highly viscous samples with dithiothreitol as the only manual step, thus offering advantages for timely availability of molecular-based detection assays. PMID:23678059
Väisänen, E; Lahtinen, A; Eis-Hübinger, A M; Lappalainen, M; Hedman, K; Söderlund-Venermo, M
Human parvovirus 4 (PARV4) of the family Parvoviridae was discovered in a plasma sample of a patient with an undiagnosed acute infection in 2005. Currently, three PARV4 genotypes have been identified, however, with an unknown clinical significance. Interestingly, these genotypes seem to differ in epidemiology. In Northern Europe, USA and Asia, genotypes 1 and 2 have been found to occur mainly in persons with a history of injecting drug use or other parenteral exposure. In contrast, genotype 3 appears to be endemic in sub-Saharan Africa, where it infects children and adults without such risk behaviour. In this study, a novel straightforward and cost-efficient molecular assay for both quantitation and genotyping of PARV4 DNA was developed. The two-step method first applies a single-probe pan-PARV4 qPCR for screening and quantitation of this relatively rare virus, and subsequently, only the positive samples undergo a real-time PCR-based multi-probe genotyping. The new qPCR-GT method is highly sensitive and specific regardless of the genotype, and thus being suitable for studying the clinical impact and occurrence of the different PARV4 genotypes.
Lebuhn, Michael; Derenkó, Jaqueline; Rademacher, Antje; Helbig, Susanne; Munk, Bernhard; Pechtl, Alexander; Stolze, Yvonne; Prowe, Steffen; Schwarz, Wolfgang H.; Schlüter, Andreas; Liebl, Wolfgang; Klocke, Michael
Five institutional partners participated in an interlaboratory comparison of nucleic acid extraction, RNA preservation and quantitative Real-Time PCR (qPCR) based assays for biogas biocenoses derived from different grass silage digesting laboratory and pilot scale fermenters. A kit format DNA extraction system based on physical and chemical lysis with excellent extraction efficiency yielded highly reproducible results among the partners and clearly outperformed a traditional CTAB/chloroform/isoamylalcohol based method. Analytical purpose, sample texture, consistency and upstream pretreatment steps determine the modifications that should be applied to achieve maximum efficiency in the trade-off between extract purity and nucleic acid recovery rate. RNA extraction was much more variable, and the destination of the extract determines the method to be used. RNA stabilization with quaternary ammonium salts was an as satisfactory approach as flash freezing in liquid N2. Due to co-eluted impurities, spectrophotometry proved to be of limited value for nucleic acid qualification and quantification in extracts obtained with the kit, and picoGreen® based quantification was more trustworthy. Absorbance at 230 nm can be extremely high in the presence of certain chaotropic guanidine salts, but guanidinium isothiocyanate does not affect (q)PCR. Absolute quantification by qPCR requires application of a reliable internal standard for which correct PCR efficiency and Y-intercept values are important and must be reported. PMID:28952569
Frølund, Maria; Björnelius, Eva; Lidbrink, Peter; Ahrens, Peter; Jensen, Jørgen Skov
A novel multiplex quantitative real-time polymerase chain reaction (qPCR) for simultaneous detection of U. urealyticum and U. parvum was developed and compared with quantitative culture in Shepard's 10 C medium for ureaplasmas in urethral swabs from 129 men and 66 women, and cervical swabs from 61 women. Using culture as the gold standard, the sensitivity of the qPCR was 96% and 95% for female urethral and cervical swabs, respectively. In male urethral swabs the sensitivity was 89%. The corresponding specificities were 100%, 87% and 99%. The qPCR showed a linear increasing DNA copy number with increasing colour-changing units. Although slightly less sensitive than culture, this multiplex qPCR assay detecting U. urealyticum and U. parvum constitutes a simple and fast alternative to the traditional methods for identification of ureaplasmas and allows simultaneous species differentiation and quantitation in clinical samples. Furthermore, specimens overgrown by other bacteria using the culture method can be evaluated in the qPCR. PMID:25047036
Ogorzaly, Leslie; Bonot, Sébastien; Moualij, Benaissa El; Zorzi, Willy; Cauchie, Henry-Michel
Development of rapid, sensitive and specific methods for detection of infectious enteric viruses in water is challenging but crucial for gaining reliable information for risk assessment. An immunocapture real-time PCR (IC-qPCR) was designed to detect jointly the two major viral particle components, i.e. the capsid protein and the viral genome. Targeting both constituents helps circumventing the technical limits of cell culture approaches and the inability of PCR based methods to predict the infectious status. Two waterborne pathogenic virus models, human adenovirus types 2 and 41, were chosen for this study. IC-qPCR showed a detection limit of 10MPNCU/reaction with a dynamic range from 10(2) to 10(6)MPNCU/reaction. Sensitivity was thus 100-fold higher compared to ELISA-based capture employing the same anti-hexon antibodies. After optimisation, application on environmental water samples was validated, and specificity towards the targeted virus types was obtained through the qPCR step. Heat-treated pure samples as well as surface water samples brought evidence that this method achieves detection of encapsidated viral genomes while excluding free viral genome amplification. As a consequence, adenovirus concentrations estimated by IC-qPCR were below those calculated by direct qPCR. The results demonstrate that the IC-qPCR method is a sensitive and rapid tool for detecting, in a single-tube assay, structurally intact and thus potentially infectious viral particles in environmental samples.
Nauwelaers, David; Vijgen, Leen; Atkinson, Claire; Todd, Alison; Geretti, Anna Maria; Van Ranst, Marc; Stuyver, Lieven
Elderly infected with Human Respiratory Syncytial Virus (RSV) often bear low viral loads that stay below the detection limits of commercial assays. A more sensitive detection of RSV infections can improve patient management, guide containment strategies, and possibly prevent morbidity and mortality among populations most severely affected by RSV. To test the sensitivity for RSV detection by using an alternative extraction method in combination with a new amplification procedure. Nasopharyngeal washes and sputum samples (n=78) form clinical cases, and broncheo-alveolar lavages (n=27) from an experimental RSV rat model were obtained. An ultrasone-based RNA extraction method was combined with a multi-component Nucleic Acid enzymes (MNAzyme) amplification procedure for simultaneous detection of RSV-A, RSV-B, and an Internal Extraction control IEC. Compared to standard real-time PCR technology, this method resulted in an increased detection sensitivity, ranging from 0.9 to 4.93 log (average 2.05+/-1.01) for RSV-A and 0.76 to 4.28 log (average 1.30+/-0.92) for RSV-B. An ultrasone-based extraction method with MNAzyme amplification resulted in improved detection of RSV in different respiratory samples, including sputum. This generic method for nucleic acid extraction should be readily applicable for any other respiratory pathogen.
Wadhwa, Ashutosh; Wilkins, Kimberly; Gao, Jinxin; Condori Condori, Rene Edgar; Gigante, Crystal M; Zhao, Hui; Ma, Xiaoyue; Ellison, James A; Greenberg, Lauren; Velasco-Villa, Andres; Orciari, Lillian; Li, Yu
Rabies, resulting from infection by Rabies virus (RABV) and related lyssaviruses, is one of the most deadly zoonotic diseases and is responsible for up to 70,000 estimated human deaths worldwide each year. Rapid and accurate laboratory diagnosis of rabies is essential for timely administration of post-exposure prophylaxis in humans and control of the disease in animals. Currently, only the direct fluorescent antibody (DFA) test is recommended for routine rabies diagnosis. Reverse-transcription polymerase chain reaction (RT-PCR) based diagnostic methods have been widely adapted for the diagnosis of other viral pathogens, but there is currently no widely accepted rapid real-time RT-PCR assay for the detection of all lyssaviruses. In this study, we demonstrate the validation of a newly developed multiplex real-time RT-PCR assay named LN34, which uses a combination of degenerate primers and probes along with probe modifications to achieve superior coverage of the Lyssavirus genus while maintaining sensitivity and specificity. The primers and probes of the LN34 assay target the highly conserved non-coding leader region and part of the nucleoprotein (N) coding sequence of the Lyssavirus genome to maintain assay robustness. The probes were further modified by locked nucleotides to increase their melting temperature to meet the requirements for an optimal real-time RT-PCR assay. The LN34 assay was able to detect all RABV variants and other lyssaviruses in a validation panel that included representative RABV isolates from most regions of the world as well as representatives of 13 additional Lyssavirus species. The LN34 assay was successfully used for both ante-mortem and post-mortem diagnosis of over 200 clinical samples as well as field derived surveillance samples. This assay represents a major improvement over previously published rabies specific RT-PCR and real-time RT-PCR assays because of its ability to universally detect RABV and other lyssaviruses, its high
Wadhwa, Ashutosh; Wilkins, Kimberly; Gao, Jinxin; Condori Condori, Rene Edgar; Gigante, Crystal M.; Zhao, Hui; Ma, Xiaoyue; Ellison, James A.; Greenberg, Lauren; Velasco-Villa, Andres; Orciari, Lillian
Rabies, resulting from infection by Rabies virus (RABV) and related lyssaviruses, is one of the most deadly zoonotic diseases and is responsible for up to 70,000 estimated human deaths worldwide each year. Rapid and accurate laboratory diagnosis of rabies is essential for timely administration of post-exposure prophylaxis in humans and control of the disease in animals. Currently, only the direct fluorescent antibody (DFA) test is recommended for routine rabies diagnosis. Reverse-transcription polymerase chain reaction (RT-PCR) based diagnostic methods have been widely adapted for the diagnosis of other viral pathogens, but there is currently no widely accepted rapid real-time RT-PCR assay for the detection of all lyssaviruses. In this study, we demonstrate the validation of a newly developed multiplex real-time RT-PCR assay named LN34, which uses a combination of degenerate primers and probes along with probe modifications to achieve superior coverage of the Lyssavirus genus while maintaining sensitivity and specificity. The primers and probes of the LN34 assay target the highly conserved non-coding leader region and part of the nucleoprotein (N) coding sequence of the Lyssavirus genome to maintain assay robustness. The probes were further modified by locked nucleotides to increase their melting temperature to meet the requirements for an optimal real-time RT-PCR assay. The LN34 assay was able to detect all RABV variants and other lyssaviruses in a validation panel that included representative RABV isolates from most regions of the world as well as representatives of 13 additional Lyssavirus species. The LN34 assay was successfully used for both ante-mortem and post-mortem diagnosis of over 200 clinical samples as well as field derived surveillance samples. This assay represents a major improvement over previously published rabies specific RT-PCR and real-time RT-PCR assays because of its ability to universally detect RABV and other lyssaviruses, its high
Lin, Yiman; Shi, Xiaolu; Zhu, Yumei; Qiu, Yaqun; Li, Yinhui; Lv, Dongyue; Hu, Qinghua
To develop real-time PCR assay based on modified molecular beacon for simultaneous detection of S. choleraesuis and S. paratyphi C. The established method was applied to the rapid detection of S. choleraesuis in food and stool samples of food poisoning, and then was applied to the identification of Salmonella C. Based on the sequences (CP000857.1) published in GenBank, Two sets of primers and modified molecular beacon were designed. The Real-time PCR assay for the simultaneous detection of S. paratyphi C and S. choleraesuis was developed with optimized PCR procedures and PCR components, while other 11 different bacterial species were as the control. Then the sensitivity and specificity of the assay were tested using 77 Samonella strains. The assay was applied to the detection of 70 food samples. The limit of detection achieved was 10 fg/reaction or 20 CUF/reaction, Only Salmonella paratyphi C and Salmonella choleraesuis strains generated fluorescent signals. No cross-reaction was observed with other 11 bacterium, the sensitivity and specificity were both 100%. No samples among 70 food samples were found Salmonella positive by both real-time PCR assay and traditional culture method. It could be finished within 2 hours from template preparation to detection and the overall test would be finished within one day. The real-time PCR assay was rapid, sensitive and specific. It could be applied to the rapid diagnosis of S. paratyphi C and S. choleraesuis in food and stool samples of food poisoning and the identification of Salmonella C to guarantee food safety.
Das, Amaresh; Deng, Ming Y; Babiuk, Shawn; McIntosh, Michael T
Capripoxviruses (CaPVs), consisting of Sheeppox virus (SPV), Goatpox virus (GPV), and Lumpy skin disease virus (LSDV) species, cause economically significant diseases in sheep, goats, and cattle, respectively. Quantitative real-time polymerase chain reaction (qPCR) assays are routinely used for rapid detection of CaPVs in surveillance and outbreak management programs. We further modified and optimized 2 previously published CaPV qPCR assays, referred to as the Balinsky and Bowden assays, by changing commercial PCR reagents used in the tests. The modified assays displayed 100% analytical specificity and showed no apparent changes in analytical sensitivities for detection of CaPVs compared with the original assays. Diagnostic sensitivities, assessed using 50 clinical reference samples from experimentally infected sheep, goats, and cattle, improved from 82% to 92% for the modified Balinsky assay and from 58% to 82% for the modified Bowden assay. The modified qPCR assays were multiplexed for detection of beta-actin as an indicator for potential false-negative results. The multiplex modified qPCR assays exhibited the same diagnostic sensitivities as the singleplex assays suggesting their utility in the detection of CaPVs.
Hasan, Mohammad R; Tan, Rusung; Al-Rawahi, Ghada; Thomas, Eva; Tilley, Peter
Quantitative, viral load monitoring for BK virus (BKV) by real-time PCR is an important tool in the management of polyomavirus associated nephropathy in renal transplant patients. However, variability in PCR results has been reported because of polymorphisms in viral genes among different subtypes of BKV, and lack of standardization of the PCR assays among different laboratories. In this study we have compared the performance of several laboratory developed PCR assays that target highly conserved regions of BKV genome with a commercially available, RealStar(®) BKV PCR Kit. Three real-time PCR assays (i) VP1 assay: selected from the literature that targets the major capsid protein (VP1) gene (ii) VP1MOD assay: VP1 assay with a modified probe, and (iii) BKLTA assay: newly designed assay that targets the large T antigen gene were assessed in parallel, using controls and clinical specimens that were previously tested using RealStar(®) BKV PCR Kit (Altona Diagnostics GmbH, Hamburg, Germany). Nucleic acid from all samples were extracted using the QIA symphony virus/bacteria kit on an automated DNA extraction platform QIA symphony SP (Qiagen). Primer and probe concentration, and reaction conditions for laboratory developed assays were optimized and the limit of detection of different assays was determined. Positive control for laboratory developed BK assays was prepared through construction of a plasmid carrying respective amplicon sequences. The 95% detection limit of VP1, VP1MOD and BKLTA assays were 1.8×10(2), 3×10(3) and 3.5×10(2) genomic copies/ml, respectively, as determined by Probit regression analysis of data obtained by testing a dilution series of a titered patient specimen, using RealStar(®) BKV PCR Kit. The inter-assay and intra-assay, coefficient of variations of these assays using calibrated, plasmid standards were <1%. All assays, including the RealStar(®) BKV PCR assay, were highly specific when tested against a panel of external proficiency
Vermehren, Johannes; Susser, Simone; Berger, Annemarie; Perner, Dany; Peiffer, Kai-Henrik; Allwinn, Regina; Zeuzem, Stefan; Sarrazin, Christoph
Virologic response-monitoring is essential for determining therapy duration in patients with chronic hepatitis C virus (HCV) infection. This is usually performed using highly sensitive HCV-RNA assays. However, HCV-RNA assays are time-consuming, expensive and require highly trained personnel. Quantitative determination of HCV core-antigen (HCVAg) levels may be used to supplement treatment monitoring. The clinical utility of the ARCHITECT HCV Ag assay (Abbott Diagnostics) for response-guided therapy was investigated. We analyzed serum from 160 patients with HCV genotype 1 infection who had been treated with peg-interferon alfa-2b/ribavirin. HCVAg levels were determined at baseline, weeks 1, 2, 4 and 12. HCVAg levels were compared to those obtained with HCV-RNA assays: VERSANT HCV Quantitative 3.0 (bDNA) and Qualitative (TMA, both Siemens Healthcare) assay and the Abbott RealTime HCV assay (ART; Abbott Diagnostics). Baseline HCVAg levels correlated well with HCV-RNA as assessed by bDNA (r=0.91; p<0.0001) and ART (r=0.92; p<0.0001), respectively. Patients with undetectable HCVAg levels at week 1 had a 90.9% probability (positive predictive value) to achieve a rapid virologic response (HCV-RNA undetectable at week 4) based on TMA and 86.4% based on ART, respectively. Patients with less than 1 log(10) reduction in HCVAg between baseline and week 12 had a 90% probability (negative predictive value) to achieve a nonresponse (<2 log(10) decline in HCV-RNA between baseline and week 12) based on bDNA and 100% based on ART, respectively. Determination of HCVAg may be useful for antiviral response-monitoring in patients with HCV genotype 1 infection. Copyright © 2012 Elsevier B.V. All rights reserved.
Lu, Rou-jian; Zhang, Ling-lin; Tan, Wen-jie; Zhou, Wei-min; Wang, Zhong; Peng, Kun; Ruan, Li
We designed specific primers and fluorescence-labeled probes to develop real-time and conventional RT-PCR assays for detection of human coronavirus NL63 or HKU1. Subsequently, experiments were undertaken to assess diagnostic criteria such as specificity, sensitivity and reproducibility. The detection limit of the real-time RT-PCR assays was 10 RNA copies per reaction mixture. No cross-reactivity was observed between RNA samples derived from designed HCoV and other HCoV or human metapneumovirus. A total of 158 nasopharyngeal swab specimens collected from adult patients with acute respiratory tract infection in Beijing were screened for the presence of human coronavirus NL63 and HKU1 by using real-time RT-PCR and conventional RT-PCR method. The fluorescence quantitative RT-PCR method detected six specimens positive for human coronavirus NL63, five specimens positive for human coronavirus HKU1; and conventional RT-PCR method detected three HCoV-NL63 positive and three HCoV-HKU1 positive, respectively. The convention RT-PCR products of positive samples were obtained and sequence analysis confirmed the reliability of the above methods. In summary, the real-time RT-PCR assay for HCoV- NL63 or HKU1 was more sensitive than conventional RT-PCR and with less time (less than 4 hours) for completion. It may be suitable for molecular epidemiological surveillance and clinical diagnosis for human coronavirus NL63 and HKU1.
Kim, Sunghyun; Kim, Young Keun; Lee, Hyejon; Cho, Jang-Eun; Kim, Hyo Youl; Uh, Young; Kim, Young Mi; Kim, Hyunjung; Cho, Sang-Nae; Jeon, Bo-Young; Lee, Hyeyoung
The interferon gamma (IFN-γ) release assay (IGRA) is widely used as a diagnostic method for latent tuberculosis infection (LTBI). The QuantiFERON-TB Gold and QuantiFERON-TB Gold In-tube (QFT-IT) tests measure plasma IFN-γ levels using enzyme-linked immunosorbent assay (ELISA), and T-SPOT.TB counts IFN-γ-producing cells using enzyme-linked immunosorbent spot assay. IFN-γ mRNA was evaluated as an indicator of IGRA in comparison with QFT-IT IFN-γ ELISA in 46 subjects with active TB and in 73 at low risk for TB. Significant IFN-γ mRNA expression was detected from 30 min and peaked 4 h after stimulation with MTB antigens or mitogen. This was defined as the optimal time point for IFN-γ mRNA real-time polymerase chain reaction (PCR). The sensitivities of IFN-γ mRNA real-time PCR and IFN-γ ELISA were 84.8% (39/46) and 89.1% (41/46), respectively (no significant difference). Although the specificities of IFN-γ ELISA was 4.1% higher than that of IFN-γ mRNA real-time PCR (60.3% versus 56.2%), the difference was not statistically significant. The overall agreement between IFN-γ mRNA real-time PCR and IFN-γ ELISA was 79.8% (kappa = 0.475). Whilst there was no difference in the performance of IFN-γ mRNA real-time PCR and IFN-γ ELISA, IFN-γ mRNA real-time PCR was superior to IFN-γ ELISA in terms of the time required for detection of MTB infection. Copyright © 2013 Elsevier Inc. All rights reserved.
Qin, Yu; Zhang, Hongyun; Marlowe, Natalia; Fei, Mandong; Yu, Judy; Lei, Xiaoqin; Yu, Lulu; Zhang, Jia; Cao, Di; Ma, Li; Chen, Wen
HIV+/AIDS women have an increased risk of developing into CIN and cervical cancer compared to the general population. Limited medical resource and the lack of AIDS relevant knowledge impair the coverage and efficiency of cervical cancer screening. To compare the clinical performance of self-collected dry storage medium (FTA Elute card) and physician-collected PreservCyt medium in detection of high risk human papillomavirus (HR HPV) among HIV-1 positive population. Three hundred HIV-1 positive women (aged 25-65) were recruited from Yunnan infectious hospital. Two cervicovaginal samples were collected from each participant: one was collected by the women themselves and applied on a FTA Elute card; the other one was collected by a physician and stored in PreservCyt solution. All the samples were tested for 14 HR HPV using Abbott RealTime High Risk HPV assay. Biopsies were taken for histological diagnosis if any abnormal impression was noticed under colposcopy. 291 (97.0%) of participants were eligible for this study. 101 (34.70%) participants were found HR HPV positive in both FTA card and PreservCyt samples, and 19 (6.53%) women were diagnosed as CIN2+. The HR HPV positive rate on samples collected by FTA Elute card and PreservCyt solution was 42.61% and 39.86%, respectively. The overall agreement was 87% (kappa=0.731) between FTA card and PreservCyt. The clinical sensitivity and specificity of FTA card and PreservCyt were 100%, 61.39% and 100%, 64.33%, respectively. In this study, FTA Elute card demonstrated a good performance on self-collected sample for HR HPV detection in HIV-1 positive population. For the women from low-resource area with HIV-1 infection, FTA Elute card could be an attractive sample collection method for cervical cancer screening. Copyright Â© 2016 Elsevier B.V. All rights reserved.
Background Ovine footrot is a contagious disease with worldwide occurrence in sheep. The main causative agent is the fastidious bacterium Dichelobacter nodosus. In Scandinavia, footrot was first diagnosed in Sweden in 2004 and later also in Norway and Denmark. Clinical examination of sheep feet is fundamental to diagnosis of footrot, but D. nodosus should also be detected to confirm the diagnosis. PCR-based detection using conventional PCR has been used at our institutes, but the method was laborious and there was a need for a faster, easier-to-interpret method. The aim of this study was to develop a TaqMan-based real-time PCR assay for detection of D. nodosus and to compare its performance with culturing and conventional PCR. Methods A D. nodosus-specific TaqMan based real-time PCR assay targeting the 16S rRNA gene was designed. The inclusivity and exclusivity (specificity) of the assay was tested using 55 bacterial and two fungal strains. To evaluate the sensitivity and harmonisation of results between different laboratories, aliquots of a single DNA preparation were analysed at three Scandinavian laboratories. The developed real-time PCR assay was compared to culturing by analysing 126 samples, and to a conventional PCR method by analysing 224 samples. A selection of PCR-products was cloned and sequenced in order to verify that they had been identified correctly. Results The developed assay had a detection limit of 3.9 fg of D. nodosus genomic DNA. This result was obtained at all three laboratories and corresponds to approximately three copies of the D. nodosus genome per reaction. The assay showed 100% inclusivity and 100% exclusivity for the strains tested. The real-time PCR assay found 54.8% more positive samples than by culturing and 8% more than conventional PCR. Conclusions The developed real-time PCR assay has good specificity and sensitivity for detection of D. nodosus, and the results are easy to interpret. The method is less time-consuming than either
Li, Dongmei; Fan, Qing-Hai; Waite, David W; Gunawardana, Disna; George, Sherly; Kumarasinghe, Lalith
Spider mites of the genus Tetranychus are difficult to identify due to their limited diagnostic characters. Many of them are morphologically similar and males are needed for species-level identification. Tetranychus urticae is a common interception and non-regulated pest at New Zealand's borders, however, most of the intercepted specimens are females and the identification was left at Tetranychus sp. Consequently, the shipments need to be fumigated. DNA sequencing and PCR-restriction fragment length polymorphism (PCR-RFLP) protocols could be used to facilitate the accurate identification. However, in the context of border security practiced in New Zealand, insect identifications are required to be provided within four hours of receiving the samples; thus, those molecular methods are not sufficient to meet this requirement. Therefore, a real-time PCR TaqMan assay was developed for identification of T. urticae by amplification of a 142 bp Internal Transcribed Spacer (ITS) 1 sequence. The developed assay is rapid, detects all life stages of T. urticae within three hours, and does not react with closely related species. Plasmid DNA containing ITS1 sequence of T. uritcae was serially diluted and used as standards in the real-time PCR assay. The quantification cycle (Cq) value of the assay depicted a strong linear relationship with T. urticae DNA content, with a regression coefficient of 0.99 and efficiency of 98%. The detection limit was estimated to be ten copies of the T. urticae target region. The assay was validated against a range of T. urticae specimens from various countries and hosts in a blind panel test. Therefore the application of the assay at New Zealand will reduce the unnecessary fumigation and be beneficial to both the importers and exporters. It is expected that the implementation of this real-time PCR assay would have wide applications in diagnostic and research agencies worldwide.
Li, Dongmei; Fan, Qing-Hai; Waite, David W.; Gunawardana, Disna; George, Sherly; Kumarasinghe, Lalith
Spider mites of the genus Tetranychus are difficult to identify due to their limited diagnostic characters. Many of them are morphologically similar and males are needed for species-level identification. Tetranychus urticae is a common interception and non-regulated pest at New Zealand’s borders, however, most of the intercepted specimens are females and the identification was left at Tetranychus sp. Consequently, the shipments need to be fumigated. DNA sequencing and PCR-restriction fragment length polymorphism (PCR-RFLP) protocols could be used to facilitate the accurate identification. However, in the context of border security practiced in New Zealand, insect identifications are required to be provided within four hours of receiving the samples; thus, those molecular methods are not sufficient to meet this requirement. Therefore, a real-time PCR TaqMan assay was developed for identification of T. urticae by amplification of a 142 bp Internal Transcribed Spacer (ITS) 1 sequence. The developed assay is rapid, detects all life stages of T. urticae within three hours, and does not react with closely related species. Plasmid DNA containing ITS1 sequence of T. uritcae was serially diluted and used as standards in the real-time PCR assay. The quantification cycle (Cq) value of the assay depicted a strong linear relationship with T. urticae DNA content, with a regression coefficient of 0.99 and efficiency of 98%. The detection limit was estimated to be ten copies of the T. urticae target region. The assay was validated against a range of T. urticae specimens from various countries and hosts in a blind panel test. Therefore the application of the assay at New Zealand will reduce the unnecessary fumigation and be beneficial to both the importers and exporters. It is expected that the implementation of this real-time PCR assay would have wide applications in diagnostic and research agencies worldwide. PMID:26147599
Metzgar, David; Skochko, Greg; Gibbins, Carl; Hudson, Nolan; Lott, Lisa; Jones, Morris S
In 2007, the Centers for Disease Control and Prevention (CDC) reported that Human adenovirus type 14 (HAdV-14) infected 106 military personnel and was responsible for the death of one U.S. soldier at Lackland Air Force Base in Texas. Identification of the responsible adenovirus, which had not previously been seen in North America and for which rapid diagnostic tools were unavailable, required retrospective analysis at reference laboratories. Initial quarantine measures were also reliant on relatively slow traditional PCR analysis at other locations. To address this problem, we developed a real-time PCR assay that detects a 225 base pair sequence in the HAdV-14a hexon gene. Fifty-one oropharyngeal swab specimens from the Naval Health Research Center, San Diego, CA and Advanced Diagnostic Laboratory, Lackland AFB, TX were used to validate the new assay. The described assay detected eight of eight and 19 of 19 confirmed HAdV-14a clinical isolates in two separate cohorts from respiratory disease outbreaks. The real-time PCR assay had a wide dynamic range, detecting from 10(2) to 10(7) copies of genomic DNA per reaction. The assay did not cross-react with other adenoviruses, influenza, respiratory syncytial virus, or common respiratory tract bacteria. The described assay is easy to use, sensitive and specific for HAdV-14a in clinical throat swab specimens, and very rapid since turnaround time is less than four hours to obtain an answer.
Moens, Britta; López, Giovanni; Adaui, Vanessa; González, Elsa; Kerremans, Lien; Clark, Daniel; Verdonck, Kristien; Gotuzzo, Eduardo; Vanham, Guido; Cassar, Olivier; Gessain, Antoine; Vandamme, Anne-Mieke; Van Dooren, Sonia
The human T-lymphotropic virus (HTLV) proviral load remains the best surrogate marker for disease progression. Real-time PCR techniques have been developed for detection and quantification of cosmopolitan HTLV type 1a (HTLV-1a) and HTLV-2. Since a growing level of diversity in subtypes and genotypes is observed, we developed a multiplex quantitative PCR for simultaneous detection, genotyping, and quantification of proviral loads of HTLV-1, 2, and 3. Our assay uses tax type-specific primers and dually labeled probes and has a dynamic range of 10(5) to 10 HTLV copies. One hundred sixty-three samples were analyzed, among which all of the different subtypes within each HTLV genotype could be detected. The performance of proviral load determination of our multiplex assay was compared with that of a previously published HTLV-1 singleplex quantitative PCR based on SYBR green detection, developed at a different institute. Linear regression analysis showed a statistically significant (P < 0.0001) and strong (r(2) = 0.87) correlation between proviral load values measured with the two distinct real-time PCR assays. In conclusion, our novel assay offers an accurate molecular diagnosis and genotyping, together with the determination of the proviral load of HTLV-infected individuals, in a single amplification reaction. Moreover, our molecular assay could offer an alternative when current available serological assays are insufficient.
Hanaki, Ken-Ichi; Ike, Fumio; Kajita, Ayako; Yasuno, Wataru; Yanagiba, Misato; Goto, Motoki; Sakai, Kouji; Ami, Yasushi; Kyuwa, Shigeru
A one-step SYBR Green I real-time RT-PCR assay was developed for the detection and quantification of a broad range of murine noroviruses (MNVs). The primer design was based on the multiple sequence alignments of 101 sequences of the open reading frame (ORF)1-ORF2 junction of MNV. The broad reactivity and quantitative capacity of the assay were validated using 7 MNV plasmids. The assay was completed within 1 h, and the reliable detection limit was 10 copies of MNV plasmid or 0.063 median tissue culture infective doses per milliliter of RAW264 cell culture-propagated viruses. The diagnostic performance of the assay was evaluated using 158 mouse fecal samples, 91 of which were confirmed to be positive. The melting curve analysis demonstrated the diversity of MNV in the samples. This is the first report of a broadly reactive one-step SYBR Green I real-time RT-PCR assay for detecting of MNVs. The rapid and sensitive performance of this assay makes it a powerful tool for diagnostic applications.
Prakash, Shantanu; Jain, Amita; Jain, Bhawana
Multiplex RT-PCR assays are widely used tools for detection of hepatitis viruses, but none of them provide quality check of sample. In the present study we developed a single-step triplex real-time polymerase chain reaction (PCR) assay for detection of Hepatitis B Virus (HBV) and Hepatitis C Virus (HCV) with sample quality check, by using β-actin as housekeeping gene. The primers and probes were self-designed and assay was standardized. Assay was also destined to quantitate copy numbers of HBV and HCV. This novel assay was sensitive, specific, and reproducible for detection of HBV and HCV in serum/plasma. The assay also detected all genotypes of HBV and HCV. The detection limit was 60 IU/mL for HBV and 20 IU/mL for HCV. This assay is the first assay developed on single-step platform for nucleic acid detection of HBV and HCV with an extra edge over all other assays by providing inbuilt check for quality of sample.
Clinical validation of 3 commercial real-time reverse transcriptase polymerase chain reaction assays for the detection of Middle East respiratory syndrome coronavirus from upper respiratory tract specimens.
Mohamed, Deqa H; AlHetheel, AbdulKarim F; Mohamud, Hanat S; Aldosari, Kamel; Alzamil, Fahad A; Somily, Ali M
Since discovery of Middle East respiratory syndrome coronavirus (MERS-CoV), a novel betacoronavirus first isolated and characterized in 2012, MERS-CoV real-time reverse transcriptase polymerase chain reaction (rRT-PCR) assays represent one of the most rapidly expanding commercial tests. However, in the absence of extensive evaluations of these assays on positive clinical material of different sources, evaluating their diagnostic effectiveness remains challenging. We describe the diagnostic performance evaluation of 3 common commercial MERS-CoV rRT-PCR assays on a large panel (n = 234) of upper respiratory tract specimens collected during an outbreak episode in Saudi Arabia. Assays were compared to the RealStar® MERS-CoV RT-PCR (Alton Diagnostics, Hamburg, Germany) assay as the gold standard. Results showed i) the TIB MolBiol® LightMix UpE and Orf1a assays (TIB MolBiol, Berlin, Germany) to be the most sensitive, followed by ii) the Anyplex™ Seegene MERS-CoV assay (Seegene, Seoul, Korea), and finally iii) the PrimerDesign™ Genesig® HCoV_2012 assay (PrimerDesign, England, United Kingdom). We also evaluate a modified protocol for the PrimerDesign™ Genesig® HCoV_2012 assay.
Qiao, Tie; Zheng, Pei-Ming; Ma, Rui-Hong; Luo, Xiao-Bing; Luo, Zhen-Liang
High prevalence of cholecystolithiasis in parts of East Asia has been postulated to be associated with Clonorchis sinensis infection. This study describes the development of a TaqMan-based real-time PCR assay for the detection of C. sinensis DNA in gallbladder bile and stone samples from patients with cholecystolithiasis. Primers and probe targeting the cytochrome c oxidase subunit 1 gene of mitochondrial DNA proved to be highly specific for C. sinensis and did not amplify other related heterogeneous DNA samples. The detection limit of this assay was 0.1 pg of adult C. sinensis genomic DNA. All of the egg-positive samples determined by microscopy yielded positive results by real-time PCR assay and that genetic testing of gallbladder stones using real-time PCR was considered as the most effective means for assessing C. sinensis infection status. This assay not only contributes to a greater understanding of stone pathogenesis but also benefits patients with cholecystolithiasis by facilitating effective diagnosis, treatment, and relapse prevention.
Landolt, Gabriele A; Karasin, Alexander I; Hofer, Christian; Mahaney, Julie; Svaren, John; Olsen, Christopher W
To evaluate sensitivity and specificity of a real-time reverse transcriptase polymerase chain reaction (RT-PCR) assay performed on pooled nasal swab specimens, compared with virus isolation performed on individual nasal swab specimens by use of 2 cell culture lines for detection of swine influenza A viruses. 900 nasal swab specimens obtained from pigs at an abattoir and 62 nasal swab specimens submitted for diagnostic testing. Primers were chosen to amplify a conserved portion of the influenza virus matrix gene. Assay sensitivity was initially determined by testing serial dilutions of various subtypes of swine influenza viruses. Sensitivity and specificity were confirmed by use of nasal swab specimens with or without addition of known concentrations of influenza virus and further validated by testing nasal swab specimens obtained through an abattoir surveillance program or submitted for diagnostic testing. Aliquots of specimens were pooled in sets of 10, and results of real-time RT-PCR assays were compared with results of virus isolation of individual specimens in Madin Darby canine kidney (MDCK) and mink lung (Mv1Lu) cells. Real-time RT-PCR assay was highly specific (100%) and sensitive (88% to 100%). Among the 16 viruses isolated, 3 grew only in Mv1Lu cells and 3 grew only in MDCK cells. Results indicate that real-time RT-PCR assay is a fast and accurate test for screening numerous nasal swab specimens for swine influenza virus. Some viruses were isolated in only MDCK or Mv1Lu cells, indicating that use of >1 cell line may be required to isolate a broad range of influenza A viruses.
Wu, Qingzhong; McFee, Wayne E; Goldstein, Tracey; Tiller, Rebekah V; Schwacke, Lori
Rapid detection of Brucella spp. in marine mammals is challenging. Microbiologic culture is used for definitive diagnosis of brucellosis, but is time consuming, has low sensitivity and can be hazardous to laboratory personnel. Serological methods can aid in diagnosis, but may not differentiate prior exposure versus current active infection and may cross-react with unrelated Gram-negative bacteria. This study reports a real-time PCR assay for the detection of Brucella spp. and application to screen clinical samples from bottlenose dolphins stranded along the coast of South Carolina, USA. The assay was found to be 100% sensitive for the Brucella strains tested, and the limit of detection was 0.27fg of genomic DNA from Brucella ceti B1/94 per PCR volume. No amplification was detected for the non-Brucella pathogens tested. Brucella DNA was detected in 31% (55/178) of clinical samples tested. These studies indicate that the real-time PCR assay is highly sensitive and specific for the detection of Brucella spp. in bottlenose dolphins. We also developed a second real-time PCR assay for rapid identification of Brucella ST27, a genotype that is associated with human zoonotic infection. Positive results were obtained for Brucella strains which had been identified as ST27 by multilocus sequence typing. No amplification was found for other Brucella strains included in this study. ST27 was identified in 33% (18/54) of Brucella spp. DNA-positive clinical samples. To our knowledge, this is the first report on the use of a real-time PCR assay for identification of Brucella genotype ST27 in marine mammals.
Fard, Somayeh Yasliani; Nomanpour, Bizhan; Fatolahzadeh, Bahram; Mobarez, Ashraf Mohebati; Darban-Sarokhalil, Davood; Fooladi, Abbas Ali Imani; Leeuwen, Willem B; Feizabadi, Mohammad Mehdi
Legionella pneumophila is an important etiological agent in both hospital and community acquired pneumonia. The sensitivity of culture for isolation of L. pneumophila from clinical specimens is low and time consuming. Similar problem also exists when the method of direct immunofluorescence is used. To detect this organism quantitatively from respiratory specimens, a Taq Man based real-time PCR targeting the mip sequence was developed. Both real-time PCR and culture methods were applied on 262 respiratory specimens from 262 ICU patients with pneumonia admitted to 5 different hospitals in Tehran. The results of real-time PCR were compared with those obtained by culture. Real-time PCR and culture found 12 and 4 specimens, respectively, as positive for L. pneumophila. Its technical specificity (100%) was checked against a panel of microorganisms consisting of both Gram-positive and Gram-negative bacteria. Our real-time PCR assay showed high sensitivity (100%) and specificity (96.9%) and could detect 200 organisms per ml from respiratory specimens. Using real-time PCR as a screening method, the frequency of nosocomial pneumonia with L. pneumophila at Tehran hospitals was estimated as 4.58%.
Kliphuis, Aletta; Bronze, Michelle; Wallis, Carole L.; Kityo, Cissy; Balinda, Sheila; Stevens, Wendy; Spieker, Nicole; de Oliveira, Tulio; Rinke de Wit, Tobias F.; Schuurman, Rob
Virological failure (VF) has been identified as the earliest, most predictive determinant of HIV-1 antiretroviral treatment (ART) failure. Due to the high cost and complexity of virological monitoring, VF assays are rarely performed in resource-limited settings (RLS). Rather, ART failure is determined by clinical monitoring and to a large extent immunological monitoring. This paper describes the development and evaluation of a low-cost, dried blood spot (DBS)-compatible qualitative assay to determine VF, in accordance with current WHO guideline recommendations for therapy switching in RLS. The assay described here is an internally controlled qualitative real-time PCR targeting the conserved long terminal repeat domain of HIV-1. This assay was applied to HIV-1 subtypes A to H and further evaluated on HIV-1 clinical plasma samples from South Africa (n = 191) and Tanzania (n = 42). Field evaluation was performed in Uganda using local clinical plasma samples (n = 176). Furthermore, assay performance was evaluated for DBS. This assay is able to identify VF for all major HIV-1 group M subtypes with equal specificity and has a lower detection limit of 1.00E+03 copies/ml for plasma samples and 5.00E+03 copies/ml for DBS. Comparative testing yielded accurate VF determination for therapy switching in 89% to 96% of samples compared to gold standards. The assay is robust and flexible, allowing for “open platform” applications and producing results comparable to those of commercial assays. Assay design enables application in laboratories that can accommodate real-time PCR equipment, allowing decentralization of testing to some extent. Compatibility with DBS extends access of sampling and thus access to this test to remote settings. PMID:23596235
Aitken, Susan C; Kliphuis, Aletta; Bronze, Michelle; Wallis, Carole L; Kityo, Cissy; Balinda, Sheila; Stevens, Wendy; Spieker, Nicole; de Oliveira, Tulio; Rinke de