Rapid Differential Detection of H1 and H3 Subtype Swine Influenza Viruses by a TaqMan-MGB Based Duplex One-Step Real Time RT-PCR Assay

Abstract Swine influenza is not only an economically important respiratory disease in swine, but also constantly poses a threat to human health. Hence, developing a rapid, sensitive and efficient detection method of swine influenza virus (SIV) is highly essential. By aligning the HA gene sequences of SIV circulating in China in recent 10 years, a H1 primer-probe set targeting both Eurasian avian-like H1N1 (EA H1N1) and Pandemic 2009 H1N1 (Pdm09 H1N1) lineages plus a H3 prime-probe set targeting the prevalent human-like H3N2 (HL H3N2) subtype were designed, respectively. Further, a TaqMan-MGB based duplex one-step real time RT-PCR (RRT-PCR) assay was established and evaluated. The duplex RRT-PCR possessed the detection limit of 5 copies/μL HA plasmid for each of the EA H1N1, Pdm09 H1N1 and HL H3N2 subtype SIVs, and matched an overall detection sensitivity of 100% and specificity of 91.67% with traditional virus isolation through chicken embryo inoculation using experimentally infected mice lung samples. Besides, the method showed high repeatability both within-run and between-runs, and no cross-reactivity against some commonly circulated porcine viruses in China. Furthermore, the duplex RRT-PCR method revealed a relatively higher prevalent rate of H1 than H3 subtype SIV in 166 nasal swabs from pigs in some slaughterhouse during October ~ December, 2019. This developed assay could be very helpful for rapid differential detection and routine surveillance of EA H1N1, Pdm09 H1N1 and HL H3N2 subtype SIVs in China.