Detection and Subtyping of Swine Influenza Viruses in Clinical Samples by the Mean of Developed Multiplex Polymerase Chain Reaction Assays

Multiplex PCR assays that can detect and identify three haemagglutinins and two neuraminidases of three main subtypes: H1N1, H1N2, and H3N2 of swine influenza virus (SIV), circulating in a pig population, were developed. Three oligonucleotide primer sets were evaluated based on the published sequences, with unique sizes characteristic for each subtype. The sequences of each primers were demonstrated to be specific for every subtype of SIV with the cDNA of reference viruses. Furthermore, the assays could detect and subtype up to 10 dilution of 10 EID50/0.2 mL of H1N1 and 10 dilution of 10 EID50 /0.2 mL of H1N2. For the H3N2 mPCR test, sensitivity was observed in a dilution as low as 10, which equals 10 EID50/0.2 mL. Conditions for the reactions and reagents concentrations were optimised. The optimal temperature was also ensemble. For all RNA positive samples in the RTnested-PCR test for influenza A viruses, the mPCR agreed completely. In 19 farms (95% of cases) the H1N1 subtype was determined, and in one farm H3N2 subtype was confirmed. Therefore, these methods could facilitate the rapid and accurate subtyping of influenza A viruses directly from field specimens.