Development of a multiplex real-time RT-PCR assay for detection of influenza A, influenza B, RSV and typing of the 2009-H1N1 influenza virus.

A high-throughput real-time RT-PCR assay was developed to amplify and detect a conserved region of the hemagglutinin gene of the 2009-H1N1 influenza A virus using a minor groove binder-conjugated hybridization probe. The assay was paired with a separate triplex real-time assay that detects influenza A via the matrix gene, influenza B and RSV in a multiplex format and compared with the Centers for Disease Control and Prevention (CDC) rRT-PCR assay using 143 samples. The 2009-H1N1 portion of the multiplex assay had 100% correlation with the CDC assay, while the triplex assay had a 99% agreement. An additional 105 samples collected from October to November 2009 were also tested using both the individual 2009-H1N1 and triplex assays. Of these 105 samples, eight were positive for the hemagglutinin target in the H1N1 assay and negative for the matrix target in the triplex assay. Discrepant analysis revealed single nucleotide polymorphisms within the matrix gene of 2009-H1N1 virus-positive samples. The limit of detection for the 2009-H1N1 assay was between 750 and 1,500 copies/reaction and no cross-reactivity with other respiratory pathogens was observed. Overall, this multiplexed format proved to be sensitive, robust and easy to use and serves as a useful tool for pandemic testing. Copyright (c) 2010 Elsevier B.V. All rights reserved.