Amplification of four genes of influenza A viruses using a degenerate primer set in a one step RT-PCR method

We designed a degenerate primer set that yielded full-length amplification of hemagglutinin (HA), neuraminidase (NA), matrix (M), and non-structural protein (NSP) genes of influenza A viruses in a single reaction mixture. These four genes were amplified from 15 HA (1-15) and 9 NA (1-9) subtypes of influenza A viruses of avian (n=16) origin. In addition, 272 field isolates of avian origin were tested by this method. Full-length amplification of HA, NA, M, and NSP genes was obtained in 242 (88.9%), 254 (93.4%), 268 (98.5%), and 268 (98.5%) isolates, respectively. No gene was amplified in four isolates. Of these four isolates, two were subtyped as H4N6, one as H7N7, and one as H10N7. Amplification was successful for all 4 genes of H1N1, H2N3, and H3N2 isolates of swine influenza. Also, all four genes were amplified in one equine influenza (H3N8) isolate and seven isolates of human origin (H1N1 and H3N2). This appears to be the first study using degenerate primer set for full-length amplification of four genes of influenza A viruses in a single reaction. Further studies are needed to determine if this primer set can be used for subtyping of influenza virus isolates.