Development and Application of Duplex Real-time RT-PCR Assay for Detection of H1 and H3 Subtype Swine Influenza Viruses

To establish a rapid, accurate method to diagnose and detect H1 and H3 subtype swine influenza viruses (SIV) at the same time, the specific primers and TaqMan probes were designed according to the conserved region of the HA gene of H1 and H3 subtype SIV. A duplex Real-time RT-PCR assay was developed for detection of H1 and H3 subtype SIV. The results showed that the Real-time RT-PCR could detect 10² copies/μL of H1 and H3 subtype SIV, the sensibility was well. Coefficient of variation of Ct value between repeating groups were all below 5%, the repeatability was favorable. The results were negative for the detection of H4, H5, H7, H9 subtypes SIV, classical swine fever virus, porcine reproductive and respiratory syndrome virus, foot and disease virus and pseudorabies virus, the specificity was fine. One sample was H1 subtype SIV, and one sample was H3 subtype SIV, by the established assay, the positive rate was 1.16%. The method was highly accurate, rapid, sensitive and specific, and could provide a method for rapid detection and epidemiological investigation of H1 and H3 subtype SIV.