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

Background 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. Objectives 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. Methods 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. Results 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. Conclusion 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.