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A matrix gene-based multiplex real-time RT-PCR for detection and differentiation of 2009 pandemic H1N1 and other influenza A viruses in North America.

ABSTRACT: The emergence in humans of pandemic (H1N1) 2009 (pH1N1) with similarities to swine influenza viruses (SIVs) caused much concern for both human and animal health as potential for interspecies transmission was initially unknown.The goal of this study was to develop a real-time RT-PCR test for the detection and differentiation of 2009 pH1N1 and endemic influenza A viruses in North America.Matrix (M) gene sequences from U.S. human pH1N1 cases and U.S. SIVs were aligned to determine a suitable region for an assay target. Primers were selected to amplify all influenza A. Two probes were designed to differentiate pH1N1 (EA matrix) from endemic (NA matrix) SIVs. The assay was validated using the first U.S. pH1N1 strain, 10 human pH1N1-positive specimens and nine U.S. SIV isolates, then evaluated on 165 specimens of swine and other animal origin submitted to the Iowa State University Veterinary Diagnostic Laboratory. Results were compared to other influenza A PCR assays. Sequences from additional pH1N1 strains and contemporary H1N1 SIVs were used to assess robustness of the selected primers and probes for the intended purpose.The new assay's results from clinical specimens concurred with confirmatory PCR testing. The additional probe designed from sequence analysis improved detection of the NA matrix subtype when added to the reaction mixture.This assay detects and differentiates pH1N1 and US influenza A viruses in various sample matrices and species. Good bioinformatics support is critical when designing RT-PCR assays and monitoring their performance.

SUBMITTER: Harmon K

PROVIDER: S-EPMC4634613 | biostudies-literature | 2010 Nov

REPOSITORIES: biostudies-literature

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A matrix gene-based multiplex real-time RT-PCR for detection and differentiation of 2009 pandemic H1N1 and other influenza A viruses in North America.

Harmon Karen K Bower Leslie L Kim Won-Il WI Pentella Michael M Yoon Kyoung-Jin KJ

Influenza and other respiratory viruses 20101001 6

<h4>Background</h4>The emergence in humans of pandemic (H1N1) 2009 (pH1N1) with similarities to swine influenza viruses (SIVs) caused much concern for both human and animal health as potential for interspecies transmission was initially unknown.<h4>Objectives</h4>The goal of this study was to develop a real-time RT-PCR test for the detection and differentiation of 2009 pH1N1 and endemic influenza A viruses in North America.<h4>Methods</h4>Matrix (M) gene sequences from U.S. human pH1N1 cases and ...[more]

PMID: 20958935

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Project description:Dengue fever results from infection with one or more of four different serotypes of dengue virus (DENV). Despite the widespread nature of this infection, available molecular diagnostics have significant limitations. The aim of this study was to develop a multiplex, real-time, reverse transcriptase-PCR (rRT-PCR) for the detection, quantitation, and serotyping of dengue viruses in a single reaction.An rRT-PCR assay targeting the 5' untranslated region and capsid gene of the DENV genome was designed using molecular beacons to provide serotype specificity. Using reference DENV strains, the assay was linear from 7.0 to 1.0 log?? cDNA equivalents/µL for each serotype. The lower limit of detection using genomic RNA was 0.3, 13.8, 0.8, and 12.4 cDNA equivalents/µL for serotypes 1-4, respectively, which was 6- to 275-fold more analytically sensitive than a widely used hemi-nested RT-PCR. Using samples from Nicaragua collected within the first five days of illness, the multiplex rRT-PCR was positive in 100% (69/69) of specimens that were positive by the hemi-nested assay, with full serotype agreement. Furthermore, the multiplex rRT-PCR detected DENV RNA in 97.2% (35/36) of specimens from Sri Lanka positive for anti-DENV IgM antibodies compared to just 44.4% (16/36) by the hemi-nested RT-PCR. No amplification was observed in 80 clinical samples sent for routine quantitative hepatitis C virus testing or when genomic RNA from other flaviviruses was tested.This single-reaction, quantitative, multiplex rRT-PCR for DENV serotyping demonstrates superior analytical and clinical performance, as well as simpler workflow compared to the hemi-nested RT-PCR reference. In particular, this multiplex rRT-PCR detects viral RNA and provides serotype information in specimens collected more than five days after fever onset and from patients who had already developed anti-DENV IgM antibodies. The implementation of this assay in dengue-endemic areas has the potential to improve both dengue diagnosis and epidemiologic surveillance.

Dataset Information

A matrix gene-based multiplex real-time RT-PCR for detection and differentiation of 2009 pandemic H1N1 and other influenza A viruses in North America.

Publications

A matrix gene-based multiplex real-time RT-PCR for detection and differentiation of 2009 pandemic H1N1 and other influenza A viruses in North America.

Similar Datasets

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