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Catsburg, Arnold, Laura van Dommelen, Vitaly Smelov, Henry J. C. de Vries, Alevtina Savitcheva, Marius Domeika, Björn Herrmann, et al. "TaqMan Assay for SwedishChlamydiatrachomatisVariant." Emerging Infectious Diseases 13, no. 9 (September 2007): 1432–34. http://dx.doi.org/10.3201/eid1309.070263.
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Watson, David E., and Baohui Li. "TaqMan Applications in Genetic and Molecular Toxicology." International Journal of Toxicology 24, no. 3 (May 2005): 139–45. http://dx.doi.org/10.1080/10915810590948299.
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Quantification of nucleic acids has become a common procedure in many toxicology laboratories. Among the technologies that accomplish this is the fluorogenic 5 ′-nuclease assay, commonly known as TaqMan. Three TaqMan applications for genetic and molecular toxicology are presented in this article: quantification of gene expression, detection of genetic polymorphisms, and quantification of chromosomal DNA deletions. Of these, quantification of gene expression is the most widely used, and established TaqMan as a benchmark technology for nucleic acid quantification. Two additional applications, polymorphism detection and quantification of DNA deletions, demonstrate the flexibility and quantitative strengths that make TaqMan so powerful, including high precision, excellent sensitivity, and broad linear dynamic range. These and similar applications improve our ability to investigate genetic and molecular dimensions of toxicological phenomena, and have promoted the widespread use of TaqMan in toxicology departments in the pharmaceutical industry. In addition to presenting these applications, the authors discuss some of the challenges of integrating TaqMan and other new technologies into the drug development process.
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Tebbs, Robert S., Yan Y. Cao, Priva Balachandran, Olga Petrauskene, and Thomas Hammack. "TaqMan Salmonella enterica Detection Kit." Journal of AOAC INTERNATIONAL 92, no. 6 (November 1, 2009): 1895–901. http://dx.doi.org/10.1093/jaoac/92.6.1895.
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Abstract Peanut butter spiked with Salmonella enterica ser. Typhimurium was prepared by an independent laboratory and sent to Applied Biosystems to determine the sensitivity and specificity of the TaqMan Salmonella enterica Detection Kit for detecting Salmonella in peanut butter. The samples were spiked at three levels: five no-spike (0 CFU/25 g); 20 low-spike (0.2 CFU/25 g); and 20 high-spike (2 CFU/25 g). They were coded to create a blind set of 45 samples. The samples were processed based on an unpaired test design that included enrichment in buffered peptone water for the candidate method and lactose broth for the reference method. In the candidate method, a 1 mL aliquot of enriched sample was extracted using PrepMan Ultra Sample Preparation Reagent; the sample was amplified on the Applied Biosystems 7500 real-time PCR system, and analyzed for detection of Salmonella using RapidFinder Version 1.0 software. All samples processed by the candidate method were confirmed by culture according to the U.S. Food and Drug Administration's Bacteriological Analytical Manual procedures. Sensitivity, specificity, and Chi-square analysis were calculated by combining candidate method results with those of the reference method that were collected by the independent laboratory. The TaqMan Salmonella enterica Detection Kit showed 40 sensitivity, 100 specificity, and a Chi-square value equal to 1.52. Chi-square analysis indicated the candidate method and the reference method were comparable. Although the candidate method sensitivity was only 40 when compared with the reference method (unpaired samples), the sensitivity was >100 when the candidate method results were compared with those of the confirmation method (same sample enrichment).
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Li, Jin, Lilin Wang, Pasi A. Jänne, and G. Mike Makrigiorgos. "Coamplification at Lower Denaturation Temperature-PCR Increases Mutation-Detection Selectivity of TaqMan-Based Real-Time PCR." Clinical Chemistry 55, no. 4 (April 1, 2009): 748–56. http://dx.doi.org/10.1373/clinchem.2008.113381.
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Abstract Background: DNA genotyping with mutation-specific TaqMan® probes (Applied Biosystems) is broadly used in detection of single-nucleotide polymorphisms but is less so for somatic mutations because of its limited selectivity for low-level mutations. We recently described coamplification at lower denaturation temperature-PCR (COLD-PCR), a method that amplifies minority alleles selectively from mixtures of wild-type and mutation-containing sequences during the PCR. We demonstrate that combining COLD-PCR with TaqMan technology provides TaqMan genotyping with the selectivity needed to detect low-level somatic mutations. Methods: Minor-groove binder-based or common TaqMan probes were designed to contain a nucleotide that matches the desired mutation approximately in the middle of the probe. The critical denaturation temperature (Tc) of each amplicon was then experimentally determined. COLD-PCR/TaqMan genotyping was performed in 2 steps: denaturation at the Tc, followed by annealing and extension at a single temperature (fast COLD-PCR). The threshold cycle was used to identify mutations on the basis of serial dilutions of mutant DNA into wild-type DNA and to identify TP53 (tumor protein p53) and EGFR [epidermal growth factor receptor (erythroblastic leukemia viral (v-erb-b) oncogene homolog, avian)] mutations in tumors. Results: COLD-PCR/TaqMan genotyping identified G>A mutations within TP53 exon 8 (codon 273 mutation hot spot) and C>T mutations within the EGFR gene (drug-resistance mutation T790M) with a selectivity improvement of 15- to 30-fold over regular PCR/TaqMan genotyping. A second round of COLD-PCR/TaqMan genotyping improved the selectivity by another 15- to 30-fold and enabled detection of 1 mutant in 2000 wild-type alleles. Use of COLD-PCR/TaqMan genotyping allowed quantitative identification of low-level TP53 and T790 mutations in colon tumor samples and in non-small-cell lung cancer cell lines treated with kinase inhibitors. Conclusions: The major improvement in selectivity provided by COLD-PCR enables the popular TaqMan genotyping method to become a powerful tool for detecting low-level mutations in clinical samples.
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Pusterla, Nicola, Jon B. Huder, Christian M. Leutenegger, Ueli Braun, John E. Madigan, and Hans Lutz. "Quantitative Real-Time PCR for Detection of Members of the Ehrlichia phagocytophila Genogroup in Host Animals and Ixodes ricinus Ticks." Journal of Clinical Microbiology 37, no. 5 (1999): 1329–31. http://dx.doi.org/10.1128/jcm.37.5.1329-1331.1999.
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A TaqMan PCR was established for identification and quantitation of members of the Ehrlichia phagocytophila group in experimentally infected cows and in Ixodes ricinus ticks. The TaqMan PCR identified a 106-bp section of the 16S rRNA gene by use of a specific fluorogenic probe and two primers. This technique was specific for members of the E. phagocytophila group, which include E. phagocytophila, Ehrlichia equi, and the agent of human granulocytic ehrlichiosis. The TaqMan system identified 10 copies of a cloned section of the 16S rRNA gene ofE. phagocytophila. The sensitivity and specificity of the TaqMan PCR were similar to those of conventional nested PCR. The numbers of ehrlichiae in leukocytes of the two cows experimentally infected with E. phagocytophila were measured daily by TaqMan PCR and had a course similar to that of the percentages of infected leukocytes determined daily by light microscopy. The prevalence of infected free-living ticks, which were collected from areas where bovine ehrlichiosis is endemic and from regions with sporadic occurrences of granulocytic ehrlichiosis in dogs and horses, was identical as determined by nested PCR and TaqMan PCR.
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El-Sayed, Ahmed K. A., Mohamed I. Abou-Dobara, Camelia A. Abdel-Malak, and Amira A. E. El-Badaly. "Taqman hydrolysis probe application for Escherichia coli, Salmonella enterica, and Vibrio cholerae detection in surface and drinking water." Journal of Water, Sanitation and Hygiene for Development 9, no. 3 (April 8, 2019): 492–99. http://dx.doi.org/10.2166/washdev.2019.137.
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Abstract This study explored the applicability of using TaqMan qPCR (quantitative polymerase chain reaction) for Escherichia coli, Salmonella enterica and non-virulent Vibrio cholerae detection in surface and drinking water. One hundred and twenty water samples were collected monthly (January 2017–December 2017) from the surface water (input) and drinking water (output and distribution networks) of two drinking water treatment plants (DWTPs) in Damietta County, Egypt. The distribution of the studied bacteria based on their detection by TaqMan qPCR compared with membrane filtration (MF) technique showed that the higher positive samples were detected by TaqMan qPCR. The bacterial count was totally absent in all output samples. TaqMan qPCR assay (based on sequence detection of uidA, invA, and ompW) revealed 97.96%, 99.14%, and 98.3% specificity for E. coli, S. enterica, and non-virulent V. cholerae, respectively, compared with 100% specificity for all strains when MF cultures were applied. TaqMan qPCR exhibited 100% sensitivity for all strains, while it was 91.67%, 80%, and 50% using MF cultures for E. coli, S. enterica, and non-virulent V. cholerae, respectively. In conclusion, TaqMan qPCR sensitivity makes it a useful tool for urgent fast monitoring of water contamination, especially in network samples that contain low bacterial count.
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Lee, Meng-Rui, Kuei-Pin Chung, Hao-Chien Wang, Chih-Bin Lin, Chong-Jen Yu, Jen-Jyh Lee, and Po-Ren Hsueh. "Evaluation of the Cobas TaqMan MTB real-time PCR assay for direct detection of Mycobacterium tuberculosis in respiratory specimens." Journal of Medical Microbiology 62, no. 8 (August 1, 2013): 1160–64. http://dx.doi.org/10.1099/jmm.0.052043-0.
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The Cobas TaqMan MTB assay is a real-time PCR (qPCR) kit for rapid detection of Mycobacterium tuberculosis from clinical specimens. There are, however, limited studies validating its performance. We performed a prospective study in two hospitals in Taiwan on 586 respiratory specimens. By using culture as the reference method, the sensitivity and specificity of the Cobas TaqMan MTB assay were found to be 82.7 and 96.5 %, respectively. The sensitivity of the Cobas TaqMan MTB assay in acid-fast stain-negative respiratory specimens was only 34.9 %. Five specimens from five patients were positive for M. tuberculosis by the Cobas TaqMan MTB assay but were negative for M. tuberculosis by conventional culture methods. A diagnosis of pulmonary tuberculosis (TB) was made based on clinical and radiological findings as well as the response to anti-TB treatment in these five patients. Addition of data from these five specimens with discrepant results (PCR vs culture) from patients with symptoms clinically compatible with TB increased the sensitivity of the Cobas TaqMan MTB assay to 83.1 %. The Cobas TaqMan MTB assay is a rapid identification tool with a high degree of specificity for the direct detection of M. tuberculosis in respiratory specimens. The sensitivity for detecting acid-fast smear-negative respiratory specimens, however, is low.
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Harle, Alexandre, and Jean-Louis Merlin. "Comparison of COBAS 4800 KRAS, PCR TaqMan, and PCR high-resolution melting assays for the detection of KRAS somatic mutations in formalin-fixed paraffin-embedded colorectal tumors." Journal of Clinical Oncology 30, no. 30_suppl (October 20, 2012): 103. http://dx.doi.org/10.1200/jco.2012.30.30_suppl.103.
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103 Background: Colorectal cancer (CRC) is the 2nd most common cancer with more than one million new cases diagnosed every year. From 2006 to 2008, several studies showed the importance of the KRAS oncogene in the treatment of metastatic CRC (mCRC) and response to anti-EGFR therapies like cetuximab or panitumumab. KRAS is a small G protein which can bear activating mutations in 40% cases of mCRC. Numerous methods have been described for the detection of KRAS mutations in FFPE tissues. Direct sequencing is the gold standard but the most sensitive methods are real time PCR assays based. The new COBAS4800 KRAS developed by Roche Diagnostics is a CE IVD validated assay using real time PCR and TaqMelt technology. This assay is validated for the detection of 19 KRAS somatic mutations on exon 2 (codons 12/13) and exon 3 (codon 61) in specimen containing at least 5% of mutated tumoral cells. Methods: We compared COBAS with previously validated PCR TaqMan and PCR High Resolution Melting (HRM) assays. The PCR TaqMan and HRM are a real time PCR based assays. TaqMan can detect accurately 0.5 to 1% of mutated tumoral DNA on codon 12 (G12A, G12C, G12D, G12R, G12S and G12V) and on codon 13 (G13D). HRM can detect 5 to 10% of tumoral DNA on codon 12 and 13. 80 FFPE samples of colorectal tumors resections or biopsies have been collected and processed with the 3 assays. Results have been compared using kappa statistics. Results: On 80 samples, 69 were interpretable with COBAS and TaqMan and 62 with COBAS and HRM. No statistically significant difference between COBAS and TaqMan was found (k=0.942 ; p<0.001). Only 2 discordant cases were found mainly regarding codon 61 mutations not detected with TaqMan. No statistically significant difference between COBAS and HRM was found (k=0.903 ; p<0.001). Three discordant cases were found two regarding codon 61, not detected with HRM and one G12S mutation (detected by COBAS and TaqMan). Conclusions: The 3 assays can detect accurately KRAS mutations. Less ininterpretable results were found with COBAS and TaqMan assays. A pre screening with COBAS and mutation characterization with TaqMan seem to be a good scheme for routine diagnostic of KRAS mutations.
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Vergara, Michelle J., I. P. Shamini Pushparajah, John F. Mackay, and Kerry R. Everett. "Testing new PCR primers and a TaqMan™ probe for detection of Phlyctema vagabunda syn. Neofabraea alba ." New Zealand Plant Protection 72 (July 28, 2019): 278. http://dx.doi.org/10.30843/nzpp.2019.72.309.
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Phlyctema vagabunda syn. Neofabraea alba is a fungal pathogen that causes bull’s eye rot (BER) of apples. Polymerase chain reaction (PCR) primers complementary to the inter-transcribed spacer region of ribosomal DNA (ITS) and the β-tubulin gene region, and a TaqMan™ probe assay were developed to detect this pathogen. These assays were compared in quantitative PCR (qPCR) reactions for amplification of DNA extracted from several fungal species and from apple tissue. Although the ITS and the β-tubulin primers amplified all N. alba isolates, both primers also amplified a few other fungal species. The TaqMan™ probe used with published primers for N. alba only amplified N. alba isolates. The TaqMan™ assay resulted in the lowest crossing threshold (Ct) values for DNA extracted from apple fruit, leaves, and spores collected on cellophane from eight apple orchards. The TaqMan™ results were correlated with percentage BER (%BER) in a 400-apple sample harvested from the same orchards. The TaqMan™ probe assay was the most sensitive and specific qPCR protocol tested, and Ct values showed the best correlation with %BER.
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Behrendt, Lars, Jeppe L. Nielsen, Søren J. Sørensen, Anthony W. D. Larkum, Jakob R. Winther, and Michael Kühl. "Rapid TaqMan-Based Quantification of Chlorophylld-Containing Cyanobacteria in the Genus Acaryochloris." Applied and Environmental Microbiology 80, no. 10 (March 14, 2014): 3244–49. http://dx.doi.org/10.1128/aem.00334-14.
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ABSTRACTReports of the chlorophyll (Chl)d-containing cyanobacteriumAcaryochlorishave accumulated since its initial discovery in 1996. The majority of this evidence is based on amplification of the gene coding for the 16S rRNA, and due to the wide geographical distribution of these sequences, a global distribution ofAcaryochlorisspecies was suggested. Here, we present a rapid, reliable, and cost-effective TaqMan-based quantitative PCR (qPCR) assay that was developed for the specific detection ofAcaryochlorisspecies in complex environmental samples. The TaqMan probe showed detection limits of ∼10 16S rRNA gene copy numbers based on standard curves consisting of plasmid inserts. DNA from fiveAcaryochlorisstrains, i.e., MBIC11017, CCMEE5410, HICR111A, CRS, and Awaji-1, exhibited amplification efficiencies of >94% when tested in the TaqMan assay. When used on complex natural communities, the TaqMan assay detected the presence ofAcaryochlorisspecies in four out of eight samples of crustose coralline algae (CCA), collected from temperate and tropical regions. In three out of these TaqMan-positive samples, the presence of Chldwas confirmed via high-performance liquid chromatography (HPLC), and corresponding cell estimates ofAcaryochlorisspecies amounted to 7.6 × 101to 3.0 × 103per mg of CCA. These numbers indicate a substantial contribution of Chld-containing cyanobacteria to primary productivity in endolithic niches. The new TaqMan assay allows quick and easy screening of environmental samples for the presence ofAcaryochlorisspecies and is an important tool to further resolve the global distribution and significance of this unique oxyphototroph.
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BOYAPALLE, SANDHYA, IRENE V. WESLEY, H. SCOTT HURD, and P. GOPAL REDDY. "Comparison of Culture, Multiplex, and 5′ Nuclease Polymerase Chain Reaction Assays for the Rapid Detection of Yersinia enterocolitica in Swine and Pork Products†." Journal of Food Protection 64, no. 9 (September 1, 2001): 1352–61. http://dx.doi.org/10.4315/0362-028x-64.9.1352.
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Bacteriological culture was compared with multiplex and fluorogenic (TaqMan) polymerase chain reaction (PCR) assays for the detection of attachment invasion locus (ail)-bearing Yersinia enterocolitica in market weight swine, chitterlings, and ground pork. The TaqMan assay detected 1 pg of purified Y. enterocolitica DNA, whereas conventional gel-based PCR detected 1 ng of the same. The presence of ail-bearing Y. enterocolitica was tested in pork and feces artificially inoculated with Y. enterocolitica strain NADC 5561. The sensitivity limits of culture, multiplex, and TaqMan PCR assays were 4 × 103, 4 × 102, and 0.4 CFU/g, respectively, for the artificially inoculated pork. The sensitivity limits were 4 × 102, 4 × 102, and 0.4 CFU/g, respectively, for feces after a 48-h enrichment in a Yersinia selective broth. By the culture method, Y. enterocolitica was not detected in any of the swine specimens (n = 2,403) examined. By contrast, it was detected in 48 (2%) of the swine samples screened using the multiplex PCR and in 656 (27.2%) of these samples using the TaqMan assay. Using the culture method, Y. enterocolitica was detected in 8% of chitterling samples (n = 350) and in none of the ground pork samples (n = 350). It was identified in 27% of the chitterling samples using multiplex PCR and in 79% of these samples using the TaqMan assay. Ten percent of the ground pork samples contained Y. enterocolitica, as determined by the multiplex PCR, and 38% based on the TaqMan assay. The results suggest that pork products harbor more ail-bearing Y. enterocolitica than selected organs of freshly slaughtered hogs and that the TaqMan assay is more sensitive than either the multiplex PCR or traditional culture methods.
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Weng, Xiaoduan, Louise Robin, Marie-lise Audet, Linda Hébert, Ginette Lapointe, Micheline Major, Bernard Lemieux, Guy Biron, Karl Bélanger, and Denis Soulières. "Improving the Sensitivity and the Specificity of HLA-B27 Typing by Replacing Serological Tests with a TaqMan-PCR Assay." Blood 112, no. 11 (November 16, 2008): 4720. http://dx.doi.org/10.1182/blood.v112.11.4720.4720.
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Abstract HLA-B27 is a strong diagnostic biomarker as it is found in 90– 95% of ankylosing spondylitis. Routinely, the false-positive results generated by cross-reactivity of HLA-B27 monoclonal antibodies (mAbs) with some other type of HLA-B antigens are problematic. The present study aims at improving the sensitivity and specificity of typing for HLA-B27 by using a more accurate DNA assay. A real time sequence specific primer polymerase chain reaction (PCR-SSP) is performed by using MGB TaqMan oligoprobe and an ABI PRISM™ 7500 sequence detection system. The TaqMan PCR-SSP assay is compared with Immunophenotyping by flow cytometric antigen assay (BD HLA-B27-Kit, clone GS145.2). The results are finally confirmed by sequencing (ABI PRISM ® 3100). As summarised in the table, three methods are identical for clearly positive and negative results. There is 90% concordance for borderline negative results obtained by immunophenotyping with TaqMan PCR-SSC and/or sequencing. However, only 15.4% of borderline positives obtained by immunophenotyping are confirmed as true HLA-B27 positives by sequencing as well as TaqMan PCR-SSC. TaqMan PCR-SSP DNA assay completely correlates with sequencing (gold standard) with 100% PPV and NPV, compared to a PPV of 15% and a NPV of 98% for borderline results by immunophenotyping. Conclusion: when compared with flow cytometric antigen assay, typing HLA-B27 by TaqMan PCR-SSP clearly demonstrates an advantage to better establish the genotype of the patient. Specifically, there is a marked difference for ambitious positive results from immunophenotyping. Moreover, compared to immunophenotyping, there is no need of fresh samples, can test a larger number of samples concomitantly, and reduces technical time as well as cost. N = 52 Immunophenotyping TaqMan PCR-SSP Sequencing Immuno vs Sequencing TaqMan PCR-SSP vs Sequencing Positive 11 11 11 100% 100% Negative 17 17 17 100% 100% Positive (borderline by Immunophenotyping) 13 2 2 15% 100% Negative (borderline ) by Immunophenotyping) 10 9 9 98% 100%
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Wiwanitkit, Viroj. "TaqMan Real-Time PCR Assay forCoccidioides." Medical Mycology 48, no. 4 (June 2010): 679. http://dx.doi.org/10.3109/13693780903496625.
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Solinas, Antonio, Nicola Thelwell, and Tom Brown. "Intramolecular TaqMan probes for genetic analysis." Chemical Communications, no. 19 (September 10, 2002): 2272–73. http://dx.doi.org/10.1039/b207254h.
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Kalinina, O. "Nanoliter scale PCR with TaqMan detection." Nucleic Acids Research 25, no. 10 (May 15, 1997): 1999–2004. http://dx.doi.org/10.1093/nar/25.10.1999.
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Jothikumar, Narayanan, Bonnie J. Mull, Sara V. Brant, Eric S. Loker, Jeremy Collinson, W. Evan Secor, and Vincent R. Hill. "Real-Time PCR and Sequencing Assays for Rapid Detection and Identification of Avian Schistosomes in Environmental Samples." Applied and Environmental Microbiology 81, no. 12 (April 10, 2015): 4207–15. http://dx.doi.org/10.1128/aem.00750-15.
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ABSTRACTCercarial dermatitis, also known as swimmer's itch, is an allergenic skin reaction followed by intense itching caused by schistosome cercariae penetrating human skin. Cercarial dermatitis outbreaks occur globally and are frequently associated with freshwater lakes and are occasionally associated with marine or estuarine waters where birds reside year-round or where migratory birds reside. In this study, a broadly reactive TaqMan assay targeting 18S rRNA gene (ribosomal DNA [rDNA]) sequences that was based on a genetically diverse panel of schistosome isolates representing 13 genera and 20 species (the 18S rDNA TaqMan assay) was developed. A PCR assay was also developed to amplify a 28S rDNA region for subsequent sequencing to identify schistosomes. When applied to surface water samples seeded withSchistosoma mansonicercariae, the 18S rDNA TaqMan assay enabled detection at a level of 5S. mansonicercariae in 100 liters of lake water. The 18S rDNA TaqMan and 28S rDNA PCR sequencing assays were also applied to 100-liter water samples collected from lakes in Nebraska and Wisconsin where there were reported dermatitis outbreaks. Avian schistosome DNA was detected in 11 of 34 lake water samples using the TaqMan assay. Further 28S rDNA sequence analysis of positive samples confirmed the presence of avian schistosome DNA and provided a preliminary identification of the avian schistosomes in 10 of the 11 samples. These data indicate that the broadly schistosome-reactive TaqMan assay can be effective for rapid screening of large-volume water samples for detection of avian schistosomes, thereby facilitating timely response actions to mitigate or prevent dermatitis outbreaks. Additionally, samples positive by the 18S rDNA TaqMan assay can be further assayed using the 28S rDNA sequencing assay to both confirm the presence of schistosomes and contribute to their identification.
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Gelderblom, Huub C., Sandra Menting, and Marcel G. Beld. "Clinical Performance of the New Roche Cobas® Taqman HCV Test and High Pure System for Extraction, Detection and Quantitation of HCV RNA in Plasma and Serum." Antiviral Therapy 11, no. 1 (January 2006): 95–103. http://dx.doi.org/10.1177/135965350601100105.
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We evaluated the Roche COBAS® TaqMan HCV Test For Use With The High Pure System (TaqMan HPS; Roche Diagnostics), for the extraction, detection and quantitation of hepatitis C virus (HCV) RNA in serum or plasma of HCV-infected individuals. The TaqMan HPS is a real-time PCR assay with a reported linear dynamic range of 3.0x101 to 2.0x108 HCV RNA IU/ml, and a reported lower limit of detection (LLD) of 10 IU/ml. Calculation of the HCV RNA titre is based upon an external standard curve in the presence of an internal control. Intra-assay and inter-assay variation were small in reference panel members with HCV RNA ≥100 IU/ml. Genotype performance and quantitative correlation between the TaqMan HPS and the bDNA (VERSANT® HCV 3.0 assay; Bayer Diagnostics), assessed in 59 patient samples, were good for HCV genotype 1 but poor for genotypes 2, 3 and 4. For genotypes 2, 3 and 4, values obtained from the TaqMan HPS were in general 0.5 log lower than those from the bDNA. Sensitivity was poor in low viral titre samples of genotypes 1, 2, 3 and 4. The LLD (95%) was estimated at 41 HCV RNA IU/ml for genotype 4. The TaqMan HPS underestimates HCV RNA at all levels in plasma and serum from HCV-infected individuals, and the LLD should be reconsidered. This is clinically relevant because underestimation of HCV RNA levels during therapy may lead physicians into making incorrect treatment decisions.
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Jothikumar, Narayanan, Theresa L. Cromeans, Vincent R. Hill, Xiaoyan Lu, Mark D. Sobsey, and Dean D. Erdman. "Quantitative Real-Time PCR Assays for Detection of Human Adenoviruses and Identification of Serotypes 40 and 41." Applied and Environmental Microbiology 71, no. 6 (June 2005): 3131–36. http://dx.doi.org/10.1128/aem.71.6.3131-3136.2005.
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ABSTRACT A quantitative real-time TaqMan PCR assay for detection of human adenoviruses (HAdV) was developed using broadly reactive consensus primers and a TaqMan probe targeting a conserved region of the hexon gene. The TaqMan assay correctly identified 56 representative adenovirus prototype strains and field isolates from all six adenovirus species (A to F). Based on infectious units, the TaqMan assay was able to detect as few as 0.4 and 0.004 infectious units of adenovirus serotype 2 (AdV2) and AdV41, respectively, with results obtained in less than 90 min. Using genomic equivalents, the broadly reactive TaqMan assay was able to detect 5 copies of AdV40 (which had zero mismatches with the PCR primers and probe), 8 copies of AdV41, and 350 copies of AdV3 (which had the most mismatches [seven] of any adenovirus serotype tested). For specific detection and identification of F species serotypes AdV40 and AdV41, a second real-time PCR assay was developed using fluorescence resonance energy transfer (FRET) probes that target the adenovirus fiber gene. The FRET-based assay had a detection limit of 3 to 5 copies of AdV40 and AdV41 standard DNA and was able to distinguish between AdV40 and AdV41 based on melting curve analysis. Both the TaqMan and FRET PCR assays were quantitative over a wide range of virus titers. Application of these assays for detection of adenoviruses and type-specific identification of AdV40 and AdV41 will be useful for identifying these viruses in environmental and clinical samples.
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Suryani, Dek Pueteri Dewi, Putu Sanna Yustiantara, and Sagung Chandra Yowani. "DESAIN TAQMAN PROBE SECARA IN SILICO SEBAGAI PENDETEKSI MUTASI PADA KODON 516 GEN rpoB Mycobacterium tuberculosis UNTUK METODE REAL-TIME PCR." Metamorfosa: Journal of Biological Sciences 4, no. 1 (March 31, 2017): 22. http://dx.doi.org/10.24843/metamorfosa.2017.v04.i01.p04.
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The aim of this research was to in silico design of TaqMan probe. Design of TaqMan probe were conducted using software Clone Manager Suite 6. As a template, the rpoB gene of M. tuberculosis H37Rv (accession number U12205.1) was used. The results of this research were 8 sequences such as, R516MV-2, R516MV-3, R516MV-4, R516MV-5, R516MV-7, R516MV-8, R516MV-11, R516MV-13. These sequences were met the criteria of TaqMan probe, such as length of nucleotide (23-28 nucleotide), Tm value (72ºC), %GC (50-58%), runs and repeats (?4 bases), dimer structure in accordance to the requirements and does not form hairpin structures. In addition, these sequences were met labeling criteria of TaqMan probe which are including the location of G bases and the number of G-C bases in sequences. Therefore, these sequences could be labeled by FAM (reporter) at 5' end and TAMRA (quencher) at 3' end. The conclusion of this research, the sequences were met the criteria of TaqMan probe. Therefore, it could be targeted to detect mutations at codon 516 with a change of aspartic acid into valine (GAC ? GTC) by using real-time PCR method.
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Chan, Edward L., Nick Antonishyn, Ryan McDonald, Tara Maksymiw, Peter Pieroni, Evelyn Nagle, and Greg B. Horsman. "The Use of TaqMan PCR Assay for Detection of Bordetella pertussis Infection From Clinical Specimens." Archives of Pathology & Laboratory Medicine 126, no. 2 (February 1, 2002): 173–76. http://dx.doi.org/10.5858/2002-126-0173-tuotpa.
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Abstract Objective.—The routine clinical laboratory detection of Bordetella pertussis is through culture, which can require 5 to 7 days for the bacteria to grow. Using a polymerase chain reaction (PCR) assay can shorten this detection time while increasing the sensitivity of detection with similar specificity. This study compared culture with TaqMan PCR for detection of B pertussis in clinical specimens and the turnaround time for each assay during the pertussis season. Materials and Methods.—Nasopharyngeal swabs in Regan-Lowe transport media were collected from 1556 persons who had symptoms of whooping cough or who had had contact with infected persons; the swabs were submitted for B pertussis detection during the pertussis season. A single nasopharyngeal swab from each patient was submitted for both culture and TaqMan PCR detection. Upon receipt of the specimens, the swabs were inoculated onto Regan-Lowe agar for culture and incubated for up to 7 days. The same swab was processed for PCR detection using TaqMan PCR assay. A second nested PCR was used on positive specimens for resolution purposes. The TaqMan PCR assay was performed 3 to 5 days a week, whereas the culture was performed 6 days a week. All specimens were processed on the same day or earliest possible working day for TaqMan or culture, and specimens queued for resolution by nested PCR were batched. Results.—There were a total of 275 PCR positives and 28 culture positives. After resolution with the second nested PCR, the sensitivity, specificity, positive predictive value, and negative predictive value were 100%, 97.4%, 87.6%, and 100% for TaqMan PCR and 11.6%, 100%, 100%, and 85.7% for culture. The average turnaround time for positive culture was 5.1 days, and the average turnaround time for PCR was 2.3 days. Conclusion.—The TaqMan PCR assay has superior sensitivity and shorter turnaround time over culture because it can be finished within one working day. Furthermore, the same swab can be used for culture of the bacteria for antibiotic susceptibility testing. The early detection of pertussis using TaqMan PCR assay allows early intervention on the spread of the disease and the ability to culture the bacteria from the same swab, thereby eliminating the need for a second swab and allowing for detection of antibiotic resistance.
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Jothikumar, N., A. J. da Silva, I. Moura, Y. Qvarnstrom, and V. R. Hill. "Detection and differentiation of Cryptosporidium hominis and Cryptosporidium parvum by dual TaqMan assays." Journal of Medical Microbiology 57, no. 9 (September 1, 2008): 1099–105. http://dx.doi.org/10.1099/jmm.0.2008/001461-0.
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Rapid identification of the two major species of Cryptosporidium associated with human infections, Cryptosporidium hominis and Cryptosporidium parvum, is important for investigating outbreaks of cryptosporidiosis. This study reports the development and validation of a real-time PCR TaqMan procedure for detection of Cryptosporidium species and identification of C. hominis and C. parvum in stool specimens. This procedure comprised a generic TaqMan assay targeting the 18S rRNA for sensitive detection of Cryptosporidium species, as well as two other TaqMan assays for identification of C. hominis and C. parvum. The generic Cryptosporidium species assay can be duplexed with the C. parvum-specific assay. The generic Cryptosporidium species assay was able to detect ten Cryptosporidium species and did not cross-react with a panel of ten other protozoan parasites. The generic Cryptosporidium species assay could detect 1–10 oocysts in a 300 μl stool specimen, whilst each of the species-specific TaqMan assays had detection sensitivities that were approximately tenfold higher. The 18S rRNA assay was found to detect Cryptosporidium species in 49/55 DNA extracts from stool specimens containing either C. hominis or C. parvum. The C. hominis TaqMan assay correctly identified C. hominis in 24/31 validation panel specimens containing this species. The C. parvum-specific assay correctly identified C. parvum in 21/24 validation panel specimens containing this species. This real-time PCR procedure was used to detect and identify C. hominis and C. parvum in stool specimens from outbreak investigations in the USA and Botswana, resulting in identification of C. hominis and/or C. parvum in 66/67 stool specimens shown to be positive for these species using other techniques. From the outbreak specimens tested, the TaqMan procedure was found to have a specificity of 94 %. This TaqMan PCR procedure should be a valuable tool for the laboratory diagnosis of cryptosporidiosis caused by C. hominis and C. parvum during outbreak investigations.
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Khan, Irfan A., Ishrat I. Ali, Saeed Khan, and Sagheer Ahmed. "Analytical Validation of TaqMan™ Assays on the OpenArray™ Platform Using Applied Biosystems QuantStudio™ 12K Flex for the Rapid Identification of Nail Microbiota." Advanced Gut & Microbiome Research 2022 (November 15, 2022): 1–9. http://dx.doi.org/10.1155/2022/5143833.
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Several microorganisms are known to play a role in nail infections. For the successful treatment of nail microbiota, especially fungi, there is a need for precise and robust diagnostic methods. This test utilizes real-time PCR amplification to detect the presence of a microorganism in a nail sample by amplifying the genomic DNA of the organism. Here, we described a TaqMan™-OpenArray™ nail microbiota assay that is efficient and easy-to-use for the characterization of key nail microbial targets through real-time PCR. The analytical accuracy and specificity of the nail panel were 100% and 99.90% across all assays and controls tested, respectively, and it was significantly more sensitive than culture. TaqMan™-OpenArray™ testing is fast and robust to contamination. In this paper, the main questions discussed were the replacement of culture by a broad-spectrum fungal TaqMan™-OpenArray™ testing and the implementation of TaqMan™-OpenArray™ testing into routine clinical laboratory settings.
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Walczak, Yana, Stefanie Ebert, Juergen Kaschkoetoe, Thomas Schild, Astrid Ferlinz, Ramon Goni, and Thomas Langmann. "Expression Profiling of Microglia and Macrophages Using Novel Lipidomic TaqMan® Array Cards and TaqMan Array Plates." BioTechniques 46, no. 4 (April 2009): 315–17. http://dx.doi.org/10.2144/000113146.
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Roma, Cristin, Claudia Esposito, Anna Maria Rachiglio, Raffaella Pasquale, Alessia Iannaccone, Nicoletta Chicchinelli, Renato Franco, et al. "Detection of EGFR Mutations by TaqMan Mutation Detection Assays Powered by Competitive Allele-Specific TaqMan PCR Technology." BioMed Research International 2013 (2013): 1–9. http://dx.doi.org/10.1155/2013/385087.
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Epidermal growth factor receptor (EGFR) mutations in non-small-cell lung cancer (NSCLC) are predictive of response to treatment with tyrosine kinase inhibitors. Competitive Allele-Specific TaqMan PCR (castPCR) is a highly sensitive and specific technology. EGFR mutations were assessed by TaqMan Mutation Detection Assays (TMDA) based on castPCR technology in 64 tumor samples: a training set of 30 NSCLC and 6 colorectal carcinoma (CRC) samples and a validation set of 28 NSCLC cases. The sensitivity and specificity of this method were compared with routine diagnostic techniques including direct sequencing and the EGFR Therascreen RGQ kit. Analysis of the training set allowed the identification of the threshold value for data analysis (0.2); the maximum cycle threshold (Ct=37); and the cut-off ΔCt value (7) for the EGFR TMDA. By using these parameters, castPCR technology identified both training and validation set EGFR mutations with similar frequency as compared with the Therascreen kit. Sequencing detected rare mutations that are not identified by either castPCR or Therascreen, but in samples with low tumor cell content it failed to detect common mutations that were revealed by real-time PCR based methods. In conclusion, our data suggest that castPCR is highly sensitive and specific to detect EGFR mutations in NSCLC clinical samples.
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Panicker, Gitika, and Asim K. Bej. "Real-Time PCR Detection of Vibrio vulnificus in Oysters: Comparison of Oligonucleotide Primers and Probes Targeting vvhA." Applied and Environmental Microbiology 71, no. 10 (October 2005): 5702–9. http://dx.doi.org/10.1128/aem.71.10.5702-5709.2005.
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ABSTRACT We compared three sets of oligonucleotide primers and two probes designed for Vibrio vulnificus hemolysin A gene (vvhA) for TaqMan-based real-time PCR method enabling specific detection of Vibrio vulnificus in oysters. Two of three sets of primers with a probe were specific for the detection of all 81 V. vulnificus isolates by TaqMan PCR. The 25 nonvibrio and 12 other vibrio isolates tested were negative. However, the third set of primers, F-vvh1059 and R-vvh1159, with the P-vvh1109 probe, although positive for all V. vulnificus isolates, also exhibited positive cycle threshold (CT ) values for other Vibrio spp. Optimization of the TaqMan PCR assay using F-vvh785/R-vvh990 or F-vvh731/R-vvh1113 primers and the P-vvh874 probe detected 1 pg of purified DNA and 103 V. vulnificus CFU/ml in pure cultures. The enriched oyster tissue homogenate did not exhibit detectable inhibition to the TaqMan PCR amplification of vvhA. Detection of 3 × 103 CFU V. vulnificus, resulting from a 5-h enrichment of an initial inoculum of 1 CFU/g of oyster tissue homogenate, was achieved with F-vvh785/R-vvh990 or F-vvh731/R-vvh1113 primers and P-vvh875 probe. The application of the TaqMan PCR using these primers and probe, exhibited detection of V. vulnificus on 5-h-enriched natural oysters harvested from the Gulf of Mexico. Selection of appropriate primers and a probe on vvhA for TaqMan-PCR-based detection of V. vulnificus in post-harvest-treated oysters would help avoid false-positive results, thus ensuring a steady supply of safe oysters to consumers and reducing V. vulnificus-related illnesses and deaths.
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Jasirwan, Chyntia Olivia Maurine, Irsan Hasan, Andri Sanityoso Sulaiman, and Rino Alvani Gani. "Evaluation of GeneXpert for Quantification Viral Load Hepatitis C Virus." Indonesian Journal of Gastroenterology, Hepatology, and Digestive Endoscopy 21, no. 3 (December 30, 2020): 182–87. http://dx.doi.org/10.24871/2132020182-187.
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Background: GeneXpert has been used for Mycobacterium tuberculosis testing, but is currently available for HCV RNA testing. GeneXpert assay is expected to be more accurate, efficient, and cost-effective for HCV viral load quantification. This study intended to evaluate the new method quantification of plasma HCV RNA by the GeneXpert in comparison to the Roche Cobas TaqMan 96 as standard diagnostic tools.Method: A total of 54 HCV-infected plasma samples with anti-HCV positive were examined by GeneXpert assay, followed with Cobas TaqMan 96 for quantification of HCV RNA. Correlation was performed by Pearson test (in log10) and diagnostic test by Chi-square test. Sensitivity and specificity of the GeneXpert assay measured by calculating 2x2 contingency table. Bland-Altman plot were generated to assess the mean difference between the two assays.Results: GeneXpert has strong correlation to the Roche Cobas TaqMan 96 (R=0.993; P value 0.001). GeneXpert has sensitivity of 100% (95% CI: 90–100%) and specificity of 90% (95% CI: 54.1–99.5%). The Bland Altmand plot showed that one sampel has 1 log difference with the Roche Cobas TaqMan 96 measurement result.Conclusion: There was a strong correlation in the measurement of HCV RNA by GeneXpert and moreover its assay also has an excellent overall performance compared to Cobas TaqMan 96. Thus, it can be considered as a reliable tools for HCV virological response monitoring.
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Schaad, N. W., Y. Berthier-Schaad, A. Sechler, and D. Knorr. "Detection of Clavibacter michiganensis subsp. sepedonicus in Potato Tubers by BIO-PCR and an Automated Real-Time Fluorescence Detection System." Plant Disease 83, no. 12 (December 1999): 1095–100. http://dx.doi.org/10.1094/pdis.1999.83.12.1095.
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Ring rot of potato, caused by Clavibacter michiganensis subsp. sepedonicus, is one of the most regulated diseases of potatoes world wide. The organism is often difficult to detect in symptomless tubers because of low populations and slow competitive growth on available media. Polymerase chain reaction (PCR) primers and a fluorescent probe for use in the Perkin Elmer 7700 automated real time PCR detection system (TaqMan) were designed from a C. michiganensis subsp. sepedonicus-specific genomic DNA fragment for development of a BIO-PCR assay for C. michiganensis subsp. sepedonicus in potato tubers. Results of screening the primers with strains of C. michiganensis subsp. sepedonicus and other bacteria showed the primers to be specific. A total of 30 naturally infected ring rot suspect tubers were sampled by the core extract, shaker incubation procedure and assayed by (i) plating aliquots onto agar media, (ii) classical PCR, and (iii) BIO-PCR. In all, 4 tubers were positive by agar plating and pathogenicity tests, 8 by classical TaqMan PCR, and 26 by TaqMan BIO-PCR. We conclude that BIO-PCR combined with the TaqMan automated closed detection system is a rapid, reliable method of assaying large numbers of potato tuber extracts for C. michiganensis subsp. sepedonicus. Furthermore, for a large central laboratory running large numbers of PCR assays, the high-throughput TaqMan system can reduce costs per sample over the more labor-intensive classical PCR.
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Orbayinah, S., A. Hermawan, Sismindari, and A. Rohman. "Detection of pork in meatballs using probe TaqMan Real-time Polymerase Chain Reaction." Food Research 4, no. 5 (May 19, 2020): 1563–68. http://dx.doi.org/10.26656/fr.2017.4(5).227.
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This study aimed to develop a TaqMan Real-Time Polymerase Chain Reaction method, using a novel primer for detection of pork adulteration in meatballs. The study is important as it described a TaqMan method for product adulteration analysis. TaqMan is known to have a more specific result compared to SYBR green analytical method. Assay in the study combined species-specific primers and TaqMan probes to targeting 153 bp fragment of D-loop mitochondrial region of pork. A specificity test was conducted on fresh tissues of pork, beef, chicken, wild boar, dog, and mouse. Meatballs as samples were prepared from a mixture of pork-beef and wild boar-beef with concentrations as follows: 5%, 10%, 25%, 75%, 90%, and 100%. The linearity and sensitivity of the method were performed by measuring the amplification curve from the dilution series, namely 1000, 200, 100, 10, 5, 1, and 0.5 pg/μL of DNA, extracted from 100% pork meatballs. A repeatability test was conducted as many as six repetitions on 100% pork and 100% wild boar meatballs. This study showed that mitochondrial D-loop species-specific primers and TaqMan probes could identify the DNA of pork and wild boar on the fresh tissues. Additionally, it also resulted in a threshold cycle (Ct) of 17.02 and 17.95 for pork, 22.22 for wild boar, while the negative result for others. The detection limit has shown 5 pg in the meatball formulation. The Relative Standard Deviation (RSD) of repeatability was 1.936% for pork, while 2.140% for wild boar. The developed method was also applied to analyzing commercial meatballs. A TaqMan real-time PCR analytical method using specific primer targeting on 153 bp fragment of the D-loop mitochondrial region could be applied as a standard method for identifying pork and wild boar in food samples intended for halal authentication studies
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Templer, Stefanie P., Britta Seiverth, Paul Baum, Wendy Stevens, Carole Seguin-Devaux, and Sergio Carmona. "Improved Sensitivity of a Dual-Target HIV-1 Qualitative Test for Plasma and Dried Blood Spots." Journal of Clinical Microbiology 54, no. 7 (May 18, 2016): 1877–82. http://dx.doi.org/10.1128/jcm.00128-16.
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The use of nucleic acid detection for HIV type 1 (HIV-1) detection is strongly recommended in infants <18 months of age, in whom serology is unreliable. This study evaluated the Cobas AmpliPrep/Cobas TaqMan HIV-1 Qualitative Test v2.0 (TaqMan HIV-1 Qual Test, v2.0), a dual-target total nucleic acid real-time PCR assay. The limit of detection (LOD) of the new test in plasma and dried blood spots (DBS) was determined with the 2nd International HIV-1 RNA WHO standard. The specificity of the assay was tested with EDTA plasma (n= 1,301) and DBS from HIV-negative adults (n= 1,000). The sensitivity was determined using HIV-1-positive samples (n= 169 adult EDTA plasma,n= 172 adult DBS, andn= 100 infant DBS) that included group M, subtypes A to H, CRF01_AE, CRF02_AG, and groups O and N. All positive specimens and a subset of the negative specimens were also tested with the Abbott RealTime HIV-1 Qual assay (RealTime). The LOD of the TaqMan assay was 20 copies/ml in plasma and 300 copies/ml in DBS, with specificities of 99.8% in plasma and 99.9% in DBS. The TaqMan assay results were 100% concordant with RealTime results in EDTA plasma samples and in 100 HIV-1-negative adult DBS. Among 172 HIV-1-positive DBS from adults, the TaqMan assay showed positive results for all DBS while RealTime missed five DBS with low target concentrations. Infant DBS results were 100% concordant. The improved sensitivity of the Cobas AmpliPrep/Cobas TaqMan HIV-1 Qualitative Test, v2.0, compared to current commercially available assays may enable earlier diagnosis and treatment in adults and infants. The dual-target test may ensure HIV-1 detection even if a mutation is present in one of the two target regions. The DBS sample matrix facilitates virological testing in remote areas.
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Yuan, Chiu-Chin, Raymond J. Peterson, Cheng-Dian Wang, Frederico Goodsaid, and David J. Waters. "5′ Nuclease Assays for the Loci CCR5-+/Δ32, CCR2-V64I, and SDF1-G801A Related to Pathogenesis of AIDS." Clinical Chemistry 46, no. 1 (January 1, 2000): 24–30. http://dx.doi.org/10.1093/clinchem/46.1.24.
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Abstract Background: Variations within the human genome play important roles in human disease. To study variations related to susceptibility to AIDS, we have developed 5′ nuclease assays that eliminate post-PCR molecular biology steps. Methods: TaqMan assays based on the 5′ nuclease activity of Taq polymerase and fluorescent resonance energy transfer were developed to score alleles at the biallelic loci CCR5-+/Δ32, CCR2-V64I and SDF1-G801A. For each assay, 72 samples were analyzed. Data collection and analysis were performed on the Prism 7700 Sequence Detection System. For comparison with gel electrophoresis methods, each locus was also scored on a subset of 24 samples, using restriction enzymes or single-strand conformational polymorphism (SSCP). Results: Clear allelic discrimination was obtained on each of the 72 samples for all three TaqMan assays. The TaqMan scores for the subset of 24 samples were concordant with the restriction enzyme and SSCP scores. Conclusions: Because of its simplicity, speed, and potential for automation and miniaturization, TaqMan is an excellent candidate for investigation of genetic variation in clinical, research, and forensic settings.
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Hammond, Emma, Kathryn Shaw, Benedict Carnley, Stephanie P'ng, Ian James, and Richard Herrmann. "Quantitative Determination of JAK2 V617F by TaqMan." Journal of Molecular Diagnostics 9, no. 2 (April 2007): 242–48. http://dx.doi.org/10.2353/jmoldx.2007.060125.
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Liao, Jason J. Z., and Jerry W. Lewis. "A Regression-Type Classification of TaqMan Assay." Journal of Agricultural, Biological, and Environmental Statistics 5, no. 4 (December 2000): 388. http://dx.doi.org/10.2307/1400656.
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Qiu, Geng, Ying Wang, Rong Fu, Yunshao He, Zirong Chen, and Jiachang Chen. "Development of Primer-Special TaqMan® PCR." Molecular Diagnosis & Therapy 14, no. 2 (April 2010): 123–29. http://dx.doi.org/10.1007/bf03256363.
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McGuigan, Fiona E. A., and Stuart H. Ralston. "Single nucleotide polymorphism detection:allelic discrimination using TaqMan." Psychiatric Genetics 12, no. 3 (September 2002): 133–36. http://dx.doi.org/10.1097/00041444-200209000-00003.
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Ranade, Koustubh. "High-Throughput Genotyping Using the TaqMan Assay." Current Protocols in Human Genetics 32, no. 1 (January 2002): 2.10.1–2.10.6. http://dx.doi.org/10.1002/0471142905.hg0210s32.
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TSAI, TSUNG-YU, WAN-JU LEE, YU-JU HUANG, KUANG-LO CHEN, and TZU-MING PAN. "Detection of Viable Enterohemorrhagic Escherichia coli O157 Using the Combination of Immunomagnetic Separation with the Reverse Transcription Multiplex TaqMan PCR System in Food and Stool Samples." Journal of Food Protection 69, no. 10 (October 1, 2006): 2320–28. http://dx.doi.org/10.4315/0362-028x-69.10.2320.
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Enterohemorrhagic Escherichia coli O157:H7 is an infectious pathogen and outbreaks have been reported all over the world, specifically in Australia, Canada, Japan, the United States, and in various countries in Europe and South Africa. Therefore, it is necessary to develop rapid methods to determine the target pathogens for food sanitation and disease. Three combinations of primers and probes were designed to detect and identify E. coli O157 using the TaqMan detection system which focuses on the specific genes eae, rfbO157, and stxII. Reverse transcription (RT) multiplex TaqMan PCR was carried out to accurately detect viable target cells correctly. Furthermore, the acidic pretreatment and immunomagnetic separation (IMS) of food and stool samples also improved the specificity and accuracy of the RT multiplex TaqMan PCR. The developed multiplex TaqMan PCR was effective in differentiating E. coli O157, enterovirulent E. coli, and non–E. coli pathogens from 100 strains which were isolated from clinical patients and the environment. Viable and nonviable cells were also distinguished by this assay. The pretreatment protocol, which included IMS to concentrate and purify the E. coli O157, was developed and the sensitivity of the assay was improved to 100 CFU/ml in pure culture, food, and stool samples. The TaqMan PCR assay is a rapid test for the detection of E. coli O157 in food and stool matrices. It shortens the process time and increases the specificity of the pathogens detected. This is critical for improving the safety and sanitation of our food supply.
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Stults, Jennie R., Oona Snoeyenbos-West, Barbara Methe, Derek R. Lovley, and Darrell P. Chandler. "Application of the 5′ Fluorogenic Exonuclease Assay (TaqMan) for Quantitative Ribosomal DNA and rRNA Analysis in Sediments." Applied and Environmental Microbiology 67, no. 6 (June 1, 2001): 2781–89. http://dx.doi.org/10.1128/aem.67.6.2781-2789.2001.
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ABSTRACT In this study, we report on the development of quantitative PCR and reverse transcriptase PCR assays for the 16S rRNA ofGeobacter spp. and identify key issues related to fluorogenic reporter systems for nucleic acid analyses of sediments. The lower detection limit of each assay was 5 to 50 fg of genomic DNA or ≤2 pg of 16S rRNA. TaqMan PCR spectral traces from uncontaminated, amended aquifer sediments were significantly lower (P < 0.0002) than traces for the external standard curve. We also observed a similar, significant decrease in mean quencher emissions for undiluted extracts relative to those for diluted extracts (P < 0.0001). If PCR enumerations were based solely upon the undiluted sample eluant, the TaqMan assay generated an inaccurate result even though the threshold cycle (C t ) measurements were precise and reproducible in the sediment extracts. Assay accuracy was significantly improved by employing a system of replicate dilutions and replicate analyses for both DNA and rRNA quantitation. Our results clearly demonstrate that fluorescence quenching and autofluorescence can significantly affect TaqMan PCR enumeration accuracy, with subsequent implications for the design and implementation of TaqMan PCR to sediments and related environmental samples.
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Hayden, Katherine, Kelly Ivors, Carla Wilkinson, and Matteo Garbelotto. "TaqMan Chemistry for Phytophthora ramorum Detection and Quantification, with a Comparison of Diagnostic Methods." Phytopathology® 96, no. 8 (August 2006): 846–54. http://dx.doi.org/10.1094/phyto-96-0846.
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The choice of detection method for phytopathogens can be critically important in determining the success or failure of pest regulation systems. We present an assay for Phytophthora ramorum that uses 5′ fluorogenic exonuclease (TaqMan) chemistry to detect and quantify the pathogen from diseased tissue, and include a universal primer and probe set for an internal positive control. This method is sensitive, detecting as little as 15 fg of target DNA when used in a nested design or 50 fg when used in a single round of polymerase chain reaction. None of the 17 other Phytophthora spp. tested was amplified by this assay. A comparison of the nested and non-nested TaqMan assays, and of one other nested assay, showed nested methods to be significantly more sensitive than nonnested and showed that host substrate significantly affected sensitivity of all assays. The nested TaqMan protocol was successfully field-tested; P. ramorum was detected in 255 of 874 plants in California woodlands, whereas the single-round TaqMan protocol detected significantly fewer positive samples. Finally, we documented increases in the quantity of pathogen DNA in Umbellularia californica leaves in initial stages of infection.
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Satterfield, Brent C., David A. Kulesh, David A. Norwood, Leonard P. Wasieloski, Michael R. Caplan, and Jay AA West. "Tentacle Probes™: Differentiation of Difficult Single-Nucleotide Polymorphisms and Deletions by Presence or Absence of a Signal in Real-Time PCR." Clinical Chemistry 53, no. 12 (December 1, 2007): 2042–50. http://dx.doi.org/10.1373/clinchem.2007.091488.
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AbstractBackground: False-positive results are a common problem in real-time PCR identification of DNA sequences that differ from near neighbors by a single-nucleotide polymorphism (SNP) or deletion. Because of a lack of sufficient probe specificity, post-PCR analysis, such as a melting curve, is often required for mutation differentiation.Methods: Tentacle Probes™, cooperative reagents with both a capture and a detection probe based on specific cell-targeting principles, were developed as a replacement for 2 chromosomal TaqMan–minor groove binder (MGB) assays previously developed for Yersinia pestis and Bacillus anthracis detection. We compared TaqMan-MGB probes to Tentacle Probes for SNP and deletion detection based on the presence or absence of a growth curve.Results: With the TaqMan-MGB Y. pestis yp48 assays, false-positive results for Yersinia pseudotuberculosis occurred at every concentration tested, and with the TaqMan-MGB B. anthracis gyrA assays, false-positive results occurred in 21 of 29 boil preps of environmental samples of near neighbors. With Tentacle Probes no false-positive results occurred.Conclusions: The high specificity exhibited by Tentacle Probes may eliminate melting curve analysis for SNP and deletion mutation detection, allowing the diagnostic use of previously difficult targets.
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Yanazawa, Koki, Takehito Sugasawa, Kai Aoki, Takuro Nakano, Yasushi Kawakami, and Kazuhiro Takekoshi. "Development of a gene doping detection method to detect overexpressed human follistatin using an adenovirus vector in mice." PeerJ 9 (October 20, 2021): e12285. http://dx.doi.org/10.7717/peerj.12285.
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Background Gene doping is the misuse of genome editing and gene therapy technologies for the purpose of manipulating specific genes or gene functions in order to improve athletic performance. However, a non-invasive detection method for gene doping using recombinant adenoviral (rAdV) vectors containing human follistatin (hFST) genes (rAdV<hFST>) has not yet been developed. Therefore, the aim of this study was to develop a method to detect gene doping using rAdV<hFST>. Methods First, we generated rAdV<hFST> and evaluated the overexpression of the hFST gene, FST protein, and muscle protein synthesis signaling using cell lines. Next, rAdV<hFST> was injected intravenously or intramuscularly into mice, and whole blood was collected, and hFST and cytomegalovirus promoter (CMVp) gene fragments were detected using TaqMan-quantitative polymerase chain reaction (qPCR). Finally, to confirm the specificity of the primers and the TaqMan probes, samples from each experiment were pooled, amplified using TaqMan-qPCR, and sequenced using the Sanger sequencing. Results The expression of hFST and FST proteins and muscle protein synthesis signaling significantly increased in C2C12 cells. In long-term, transgene fragments could be detected until 4 days after intravenous injection and 3 days after intramuscular injection. Finally, the Sanger sequencing confirmed that the primers and TaqMan probe specifically amplified the gene sequence of interest. Conclusions These results indicate the possibility of detecting gene doping using rAdV<hFST> using TaqMan-qPCR in blood samples. This study may contribute to the development of detection methods for gene doping using rAdV<hFST>.
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Lyon, W. J. "TaqMan PCR for Detection of Vibrio cholerae O1, O139, Non-O1, and Non-O139 in Pure Cultures, Raw Oysters, and Synthetic Seawater." Applied and Environmental Microbiology 67, no. 10 (October 1, 2001): 4685–93. http://dx.doi.org/10.1128/aem.67.10.4685-4693.2001.
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ABSTRACT Vibrio cholerae is recognized as a leading human waterborne pathogen. Traditional diagnostic testing forVibrio is not always reliable, because this bacterium can enter a viable but nonculturable state. Therefore, nucleic acid-based tests have emerged as a useful alternative to traditional enrichment testing. In this article, a TaqMan PCR assay is presented for quantitative detection of V. cholerae in pure cultures, oysters, and synthetic seawater. Primers and probe were designed from the nonclassical hemolysin (hlyA) sequence of V. choleraestrains. This probe was applied to DNA from 60 bacterial strains comprising 21 genera. The TaqMan PCR assay was positive for all of the strains of V. cholerae tested and negative for all other species of Vibrio tested. In addition, none of the other genera tested was amplified with the TaqMan primers and probe used in this study. The results of the TaqMan PCR with raw oysters and spiked with V. cholerae serotypes O1 and O139 were comparable to those of pure cultures. The sensitivity of the assay was in the range of 6 to 8 CFU g−1 and 10 CFU ml−1 in spiked raw oyster and synthetic seawater samples, respectively. The total assay could be completed in 3 h. Quantification of the Vibrio cells was linear over at least 6 log units. The TaqMan probe and primer set developed in this study can be used as a rapid screening tool for the presence ofV. cholerae in oysters and seawater without prior isolation and characterization of the bacteria by traditional microbiological methods.
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Hänggeli, Kai Pascal Alexander, Andrew Hemphill, Norbert Müller, Bernd Schimanski, Philipp Olias, Joachim Müller, and Ghalia Boubaker. "Single- and duplex TaqMan-quantitative PCR for determining the copy numbers of integrated selection markers during site-specific mutagenesis in Toxoplasma gondii by CRISPR-Cas9." PLOS ONE 17, no. 9 (September 16, 2022): e0271011. http://dx.doi.org/10.1371/journal.pone.0271011.
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Herein, we developed a single and a duplex TaqMan quantitative PCR (qPCR) for absolute quantification of copy numbers of integrated dihydrofolate reductase-thymidylate synthase (mdhfr-ts) drug selectable marker for pyrimethamine resistance in Toxoplasma gondii knockouts (KOs). The single TaqMan qPCR amplifies a 174 bp DNA fragment of the inserted mdhfr-ts and of the wild-type (WT) dhfr-ts (wtdhfr-ts) which is present as single copy gene in Toxoplasma and encodes a sensitive enzyme to pyrimethamine. Thus, the copy number of the dhfr-ts fragment in a given DNA quantity from KO parasites with a single site-specific integration should be twice the number of dhfr-ts copies recorded in the same DNA quantity from WT parasites. The duplex TaqMan qPCR allows simultaneous amplification of the 174 bp dhfr-ts fragment and the T. gondii 529-bp repeat element. Accordingly, for a WT DNA sample, the determined number of tachyzoites given by dhfr-ts amplification is equal to the number of tachyzoites determined by amplification of the Toxoplasma 529-bp, resulting thus in a ratio of 1. However, for a KO clone having a single site-specific integration of mdhfr-ts, the calculated ratio is 2. We then applied both approaches to test T. gondii RH mutants in which the major surface antigen (SAG1) was disrupted through insertion of mdhfr-ts using CRISPR-Cas9. Results from both assays were in correlation showing a high accuracy in detecting KOs with multiple integrated mdhfr-ts. Southern blot analyses using BsaBI and DraIII confirmed qPCRs results. Both TaqMan qPCRs are needed for reliable diagnostic of T. gondii KOs following CRISPR-Cas9-mediated mutagenesis, particularly with respect to off-target effects resulting from multiple insertions of mdhfr-ts. The principle of the duplex TaqMan qPCR is applicable for other selectable markers in Toxoplasma. TaqMan qPCR tools may contribute to more frequent use of WT Toxoplasma strains during functional genomics.
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Boyer, S., and J. Yang. "Measurement of gene amplification in FFPE tumor tissues by quantitative real-time PCR in comparison with FISH." Journal of Clinical Oncology 25, no. 18_suppl (June 20, 2007): 14086. http://dx.doi.org/10.1200/jco.2007.25.18_suppl.14086.
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14086 Background: Amplified oncogenes such as HER2 and EGFR have proven to be good targets for molecular therapy. Recent retrospective analyses suggest that EGFR amplification predicate better responses to EGFR kinase inhibitors. However, different methods were used to measure EGFR gene amplification and polysomy in these studies, including FISH and various real-time quantitative PCR platforms, which may have led to the different conclusions reported by these groups. In this study, we compared Taqman- QPCR with FISH in scoring EGFR and HER2 amplification and EGFR polysomy on 42 FFPE tumor samples. Methods: DNA was prepared from 42 FFPE tumor tissues of breast, colon, lung, and stomach origin. Taqman QPCR was carried out according to manufacturer’s protocol on an ABI-7900HT instrument. FISH analysis for EGFR and HER2 amplification was carried out according to Vysis standard protocols. Results: QPCR reference probes for measuring EGFR and HER2 amplification were designed against DNA fragments near the centromeric regions of chromosome 7 and chromosome 17, respectively. A QPCR probe for RNase P on chromosome 6 was designed and used as a reference for measuring EGFR polysomy. HER2 amplification was detected in ∼25% FFPE breast tumor samples by both FISH and QPCR methods, although amplification scores were consistently higher by QPCR than by FISH. EGFR amplification was rare in all samples screened but high polysomy (6 copy and above) was detected in 40% FFPE gastric tumor samples. Again there was high concordance between FISH and Taqman QPCR results. Conclusions: Our data showed high concordance between Taqman QPCR and FISH in scoring EGFR and HER2 amplification. Taqman QPCR is a highly sensitive, accurate, convenient, and economical technology for measuring EGFR and HER2 amplification and EGFR polysomy in FFPE tissues. With well validated target and reference primers/probes, Taqman QPCR provides an attractive alternative to FISH for measuring oncogene amplification for retrospective studies on archived FFPE samples and for screening patients for clinical trials. No significant financial relationships to disclose.
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Sen, Keya. "Rapid Identification of Yersinia enterocolitica in Blood by the 5′ Nuclease PCR Assay." Journal of Clinical Microbiology 38, no. 5 (2000): 1953–58. http://dx.doi.org/10.1128/jcm.38.5.1953-1958.2000.
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Yersinia enterocolitica accounts for 50% of the clinical sepsis episodes caused by the transfusion of contaminated red blood cells. A 5′ nuclease TaqMan PCR assay was developed to detectY. enterocolitica in blood. Primers and a probe based on the nucleotide sequence of the 16S rRNA gene from Y. enterocolitica were designed. Whole-blood samples were spiked with various numbers of Y. enterocolitica cells, and total chromosomal DNA was extracted. When the TaqMan PCR assay was performed, as few as six bacteria spiked in 200 μl of blood could be detected. The assay was specific and did not detect other Yersiniaspecies. The TaqMan assay is easy to perform, takes 2 h, and has the potential for use in the rapid detection of Y. enterocolitica contamination in stored blood units.
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Schweiger, B., I. Zadow, R. Heckler, H. Timm, and G. Pauli. "Application of a Fluorogenic PCR Assay for Typing and Subtyping of Influenza Viruses in Respiratory Samples." Journal of Clinical Microbiology 38, no. 4 (2000): 1552–58. http://dx.doi.org/10.1128/jcm.38.4.1552-1558.2000.
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A fluorogenic PCR-based method (TaqMan-PCR) was developed for typing and subtyping of influenza virus genomes in clinical specimens. The TaqMan-PCR employs a probe technology that exploits the endogenous 5′–3′ nuclease activity of the Taq DNA polymerase to allow direct detection of the amplicon by release of a fluorescent reporter during the PCR. Therefore, post-PCR analysis is avoided since hybridization with the fluorogenic probe and quantification of the amplified product is performed simultaneously during PCR cycling. The specificity of the method was evaluated on 86 influenza A (25 H1N1 and 61 H3N2) and 49 influenza B virus reference strains and isolates. The sensitivity of the technique was found to be at the level of 0.1 50% tissue culture infective dose. This TaqMan-PCR was applied prospectively to surveillance work by community-based sampling in Germany during the last two influenza seasons. Seven hundred five throat swabs were analyzed during the winter of 1997–1998. A total of 195 of 705 samples (28%) were positive by PCR. Influenza viruses could be isolated from 125 specimens (18%). During the 1998–1999 season, 1,840 respiratory samples were received. Influenza viruses were isolated from 281 specimens (15%) out of 525 throat swabs (29%) which were positive for influenza A or B virus by TaqMan-PCR. Further differentiation of influenza A virus-positive swabs revealed an intensive circulation of the subtype H3N2 during both seasons, 1997–1998 and 1998–1999. The TaqMan-PCR was much more sensitive than culture and revealed an excellent correlation for typing and subtyping of influenza viruses when samples were positive by both methods.
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Hashish, Amro, Avanti Sinha, Amr Mekky, Yuko Sato, Nubia Macedo, and Mohamed El-Gazzar. "Development and Validation of Two Diagnostic Real-Time PCR (TaqMan) Assays for the Detection of Bordetella avium from Clinical Samples and Comparison to the Currently Available Real-Time TaqMan PCR Assay." Microorganisms 9, no. 11 (October 27, 2021): 2232. http://dx.doi.org/10.3390/microorganisms9112232.
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Bordetella avium (BA) is one of many pathogens that cause respiratory diseases in turkeys. However, other bacterial species can easily overgrow it during isolation attempts. This makes confirming the diagnosis of BA as the causative agent of turkey coryza more difficult. Currently, there are two PCR assays for the molecular detection of BA. One is conventional gel-based PCR and the other is TaqMan real-time PCR (qPCR) assay. However, multiple pitfalls were detected in both assays regarding their specificity, sensitivity, and efficiency, which limits their utility as diagnostic tools. In this study, we developed and validated two TaqMan qPCR assays and compared their performance to the currently available TaqMan qPCR. The two assays were able to correctly identify all BA isolates and showed negative results against a wide range of different microorganisms. The two assays were found to have high efficiency with a detection limit of approximately 1 × 103 plasmid DNA Copies/mL with high repeatability and reproducibility. In comparison to the currently available TaqMan qPCR assay, the newly developed assays showed significantly higher PCR efficiencies due to superior primers and probes design. The new assays can serve as a reliable tool for the sensitive, specific, and efficient diagnosis of BA.
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Weller, S. A., J. G. Elphinstone, N. C. Smith, N. Boonham, and D. E. Stead. "Detection of Ralstonia solanacearumStrains with a Quantitative, Multiplex, Real-Time, Fluorogenic PCR (TaqMan) Assay." Applied and Environmental Microbiology 66, no. 7 (July 1, 2000): 2853–58. http://dx.doi.org/10.1128/aem.66.7.2853-2858.2000.
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ABSTRACT A fluorogenic (TaqMan) PCR assay was developed to detectRalstonia solanacearum strains. Two fluorogenic probes were utilized in a multiplex reaction; one broad-range probe (RS) detected all biovars of R. solanacearum, and a second more specific probe (B2) detected only biovar 2A. Amplification of the target was measured by the 5′ nuclease activity of Taq DNA polymerase on each probe, resulting in emission of fluorescence. TaqMan PCR was performed with DNA extracted from 42 R. solanacearum and genetically or serologically related strains to demonstrate the specificity of the assay. In pure cultures, detection of R. solanacearum to ≥102 cells ml−1 was achieved. Sensitivity decreased when TaqMan PCR was performed with inoculated potato tissue extracts, prepared by currently recommended extraction procedures. A third fluorogenic probe (COX), designed with the potato cytochrome oxidase gene sequence, was also developed for use as an internal PCR control and was shown to detect potato DNA in an RS-COX multiplex TaqMan PCR with infected potato tissue. The specificity and sensitivity of the assay, combined with high speed, robustness, reliability, and the possibility of automating the technique, offer potential advantages in routine indexing of potato tubers and other plant material for the presence of R. solanacearum.
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Abdel-Hamid, Nour H., Eman I. M. Beleta, Mohamed A. Kelany, Rania I. Ismail, Nadia A. Shalaby, and Manal H. M. Khafagi. "Validation of real-time polymerase chain reaction versus conventional polymerase chain reaction for diagnosis of brucellosis in cattle sera." January-2021 14, no. 1 (2021): 144–54. http://dx.doi.org/10.14202/vetworld.2021.144-154.
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Background and Aim: Different polymerase chain reaction (PCR) techniques have and are still being used for the direct detection of Brucella DNA in serum samples of different animal species and humans without being validated or properly validated, resulting in discrepancies. Thus, this study aimed to evaluate the diagnostic performance of the TaqMan Real- Time-PCR (RT-PCR) targeting the bcsp31 gene versus conventional PCR for the accurate diagnosis of brucellosis at the genus level in cattle sera. Materials and Methods: One hundred and eighty-four serum samples were collected from bacteriologically positive and negative cows with ages ranging from 1 to 5 years old at some infected private farms in the Nile Delta under quarantine measures as well as brucellosis free farms. These samples were classified into four groups after serological diagnosis and investigated by TaqMan RT-PCR and conventional PCR targeting the IS711 gene for Brucella DNA detection. The diagnostic performance characteristics of both PCR techniques were estimated considering the bacteriological results as a gold standard. Results: TaqMan RT-PCR revealed superiority over conventional PCR; it was able to detect Brucella DNA in 95% (67/70) and 89% (25/28) of the cattle sera samples belonging to Group 1 (serologically and bacteriologically positive) and Group 2 (serologically negative but bacteriologically positive), respectively. On evaluating the diagnostic performance, TaqMan RT-PCR showed superior diagnostic sensitivity (93.9%), diagnostic specificity (88.4%), performance index (182.3), almost perfect kappa agreement (0.825±0.042), strong positive correlation (r=0.826), high accuracy based on the receiver operating characteristic (ROC) curve, and area under the ROC curve (0.911) at p<0.05 and CI of 95%. Conclusion: A cattle serum sample is not the metric of choice for targeting Brucella genomic DNA by conventional PCR. The time-saving and rapid TaqMan RT-PCR method revealed a better diagnostic performance in the detection of Brucella DNA in cattle sera. Such performance offered by TaqMan RT-PCR may be considered a step toward the possibility of using such technology in the direct differentiation between Brucella-infected and -vaccinated cattle immunized by smooth vaccines from cattle sera using primers specific for such vaccines.
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Lalama, Christina M., Cheryl Jennings, Victoria A. Johnson, Robert W. Coombs, John E. McKinnon, James W. Bremer, Bryan R. Cobb, Gavin A. Cloherty, John W. Mellors, and Heather J. Ribaudo. "Comparison of Three Different FDA-Approved Plasma HIV-1 RNA Assay Platforms Confirms the Virologic Failure Endpoint of 200 Copies per Milliliter Despite Improved Assay Sensitivity." Journal of Clinical Microbiology 53, no. 8 (June 10, 2015): 2659–66. http://dx.doi.org/10.1128/jcm.00801-15.
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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 ObuchowskiP< 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'sP< 0.007). At a 200-copies/ml VF threshold, no differences between assays were apparent (allP> 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.
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Zübert, Christina, and Michael Kube. "Application of TaqMan Real-Time PCR for Detecting ‘Candidatus Arsenophonus Phytopathogenicus’ Infection in Sugar Beet." Pathogens 10, no. 11 (November 12, 2021): 1466. http://dx.doi.org/10.3390/pathogens10111466.
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The γ-proteobacterium ‘Candidatus Arsenophonus phytopathogenicus’ is assigned as the major pathogen of “Syndrome des basses richesses”, a sugar beet disease characterised by a reduction in the sugar content of taproots and biomass yield. Despite the economic impact of this bacteriosis, diagnostics for this important pathogen currently rely on end-point PCR detection. Herein, we introduce a TaqMan qPCR for diagnostics of the agent targeting genes encoding a heat shock protein of the Hsp20 family and mannose-6-phosphate isomerase. Quantitation with synthetic oligonucleotides as standard showed that the developed TaqMan qPCR assays enable the detection of up to 100 target copies. A comparison between the TaqMan qPCR and end-point PCR for ‘Ca. A. phytopathogenicus’ detection was carried out on 78 sugar beet samples from different locations in southern Germany. The newly developed assays enable the fast, reliable and sensitive detection of ‘Ca. A. phytopathogenicus’ in sugar beet.