Dataset Information

Application of a fluorogenic PCR assay for typing and subtyping of influenza viruses in respiratory samples.

ABSTRACT: 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.

SUBMITTER: Schweiger B

PROVIDER: S-EPMC86487 | biostudies-literature | 2000 Apr

REPOSITORIES: biostudies-literature

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Application of a fluorogenic PCR assay for typing and subtyping of influenza viruses in respiratory samples.

Schweiger B B Zadow I I Heckler R R Timm H H Pauli G G

Journal of clinical microbiology 20000401 4

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 simulta ...[more]

PMID: 10747142

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Project description:The reliable and rapid detection of viral pathogens that cause respiratory infections provide physicians several advantages in treating patients and managing outbreaks. The Luminex respiratory virus panel (RVP) assay has been shown to be comparable to or superior to culture/direct fluorescent-antibody assays (DFAs) and nucleic acid tests that are used to diagnose respiratory viral infections. We developed a multiplex asymmetric reverse transcription (RT)-PCR assay that can simultaneously differentiate all influenza A virus epidemic subtypes. The amplified products were hybridized with an electrochemical DNA sensor, and the results were automatically acquired. The limits of detection (LoDs) of both the Luminex RVP assay and the multiplex RT-PCR-electrochemical DNA sensor were 101 TCID50 for H1N1 virus and 102 TCID50 for H3N2 virus. The specificity assessment of the multiplex RT-PCR-electrochemical DNA sensor showed no cross-reactivity among different influenza A subtypes or with other non-influenza respiratory viruses. In total, 3098 respiratory tract specimens collected from padiatric patients diagnosed with pneumonia were tested. More than half (43, 53.75%) of the specimens positive for influenza A viruses could not be further subtyped using the Luminex RVP assay. Among the remaining 15 specimens that were not subtyped, not degraded, and in sufficient amounts for the multiplex RT-PCR-electrochemical DNA sensor test, all (100%) were H3N2 positive. Therefore, the sensitivity of the Luminex RVP assay for influenza A virus was 46.25%, whereas the sensitivity of the multiplex RT-PCR-electrochemical DNA sensor for the clinical H1N1 and H3N2 specimens was 100%. The sensitivities of the multiplex RT-PCR-electrochemical DNA sensor for the avian H5N1, H5N6, H9N2, and H10N8 viruses were 100%, whereas that for H7N9 virus was 85.19%. We conclude that the multiplex RT-PCR-electrochemical DNA sensor is a reliable method for the rapid and accurate detection of highly variable influenza A viruses in respiratory infections with greater detection sensitivity than that of the Luminex xTAG assay. The high mutation rate of influenza A viruses, particularly H3N2 during the 2014 to 2016 epidemic seasons, has a strong impact on diagnosis. A study involving more positive specimens from all influenza A virus epidemic subtypes is required to fully assess the performance of the assay.

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Dataset Information

Application of a fluorogenic PCR assay for typing and subtyping of influenza viruses in respiratory samples.

Publications

Application of a fluorogenic PCR assay for typing and subtyping of influenza viruses in respiratory samples.

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