Real-time Fluorescent PCR In The Rapid Detection Of H3N2, H1N1 And H1N2 Subtype Swine Influenza Virus

According to the submitted nucleotide sequences of M gene, H3N2, H1N1 gene of A/Swine/Guangdong/9/2005(H3N2), A/Swine/Guangxi/12/2005(H1N1), A/Swine/Tianjin/1/ 2007(H1N2) and the sequences reported in GenBank of different strains of H3N2, H1N1, H1N2 subtype Swine Influenza Virus, five pairs of primer (M, H3, N2, H1 and N1) and five TaqMan MGB probe were synthesized, these pairs of primer used to amplify the M, H3, and N2 genes of A/Swine/Guangdong/9/2005(H3N2) and H1, N1 genes of A/Swine/Guangxi/12/ 2005(H1N1) by RT-PCR using genome RNA as template, the ligation product of the amplified fragment with the linear vector pMD18-T was then transformed into E.coli competent cells. After identified by PCR, the recombinant plasmids pMD-M, pMD-H3, pMD-N2, pMD-H1 and pMD-N1 were sequenced. The results by DNAStar analysis showed that the cloned M, H3, N2, H1, N1 genes have above 97.5% homology to that of A/Swine/Guangdong/9/2005(H3N2) and A/Swine/Guangxi/12/2005(H1N1). A Real-time PCR was established by series of experiments including the selection and optimization of concentration of primers, probes, dNTP and Mgcl2 and parameter of cycle etc.In this study a set of degenerate primers and a TaqMan MGB probe directed to M,HA and NA gene of Swine Influenza Virus genome were synthesized and used to establish a real-time PCR method to detect and discriminate various genotype of Swine Influenza Virus from other subtypes.. After systematic optimization the method was successfully adapted to quantitate the virus load in tissue specimens and able to detect as less 100 copies genes/reaction of the virus. Further detection of 19 artificial conteracting toxic substances specimens and 30 clinical specimens by real-time RT-PCR in comparison with virus isolation showed that 100 percent of them were SIV positive in real-time PCR as well as in virus isolation after, No cross-reaction was detected against CSFV, PRRSV, PCV, TGEV and PEDV, indicating that real-time PCR is faster and more sensitive than routine PCR and virus isolation and has a promising application in diagnose of clinical suspected cases.