Duplex Real-Time Reverse Transcriptase PCR Assays for Rapid Detection and Identification of Pandemic (H1N1) 2009 and Seasonal Influenza A/H1, A/H3, and B Viruses

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Abstract
Reports of a novel influenza virus type A (H1N1), now designated by the World Health Organization as pandemic (H1N1) 2009, emerged from the United States and Mexico in April 2009. The management of the pandemic in Australia required rapid and reliable testing of large numbers of specimens for the novel influenza strain and differentiation from seasonal influenza strains. A real-time reverse transcriptase PCR (RT-PCR) assay for the detection of pandemic (H1N1) 2009 was designed and used with existing real-time RT-PCR assays for seasonal influenza viruses A and B. MS2 coliphage was added to all samples and amplified as a quality control. Three duplex RT-PCR assays, each containing two primer pairs and corresponding 5' nuclease probes, were initially evaluated on control material and stored samples and showed high sensitivity and specificity. More than 11,000 clinical samples were then tested for influenza A and B matrix gene targets and specific hemagglutinin gene targets for seasonal influenza A/H1, A/H3, and pandemic A (H1N1) 2009. Minimum sensitivities and specificities were 98.8% and 100%, respectively, for pandemic (H1N1) 2009, 81.5% and 98.9% for seasonal A/H1, and 96.3% and 99.6% for A/H3. Automated sample extraction facilitated the rapid processing of samples so that the assays allowed accurate, rapid, and cost-effective screening of large numbers of clinical samples.