Establishment of a multiplex RT-PCR assay to detect different lineages of swine H1 and H3 influenza A viruses

Classical swine H1N1, emerging European avian-like H1N1 and human-like H3N2 lineages are co-circulating in the swine population in China. The reverse transcriptase polymerase chain reaction (RT-PCR) assay is an effective method for use in influenza surveillance. In this study, a multiplex RT-PCR method was developed for simultaneous identification of hemagglutinin (HA) genes derived from the three lineages of swine influenza viruses. Three primer sets were designed and aimed specifically at HA genes of these viral lineages. The specificity of the assay showed that the established methods could efficiently differentiate the HA genes of classical swine H1N1, European avian-like H1N1, and human-like H3N2 viruses while other viruses such as classical swine fever virus, porcine reproductive and respiratory syndrome virus, pseudorabies virus, and porcine circovirus type 2, could not be detected. The assay showed a sensitivity of 1 × 10².⁵ 50% egg infectious dose for each virus lineage. The comparison of the results with those obtained from the analysis of 300 swine tracheal swab samples by means of virus isolation showed a high level of agreement. This multiplex RT-PCR method provides a rapid and specific swine influenza diagnostic tool that also has the potential for investigating the epidemiology of different lineages of swine influenza virus prevalent currently in China.