猪流感病毒H1N1、H1N2和H3N2亚型多重RT-PCR诊断方法的建立

Two multiplex RT-PCR assays specific for influenza virus H1 and H3, N1 and N2 subtypes were developed. Primers were designed from conserved regions of the HA and NA genes of swine influenza H1N1, H1N2 and H3N2 viruses according to the published sequences in GenBank and sequences from China isolates of swine influenza virus. The RT-PCR amplified successfully the HA and NA genes of H1N1, H1N2 and H3N2 subtype of swine influenza viruses. Sequence analysis of PCR products by BLAST search revealed the highest identity among viruses of the same HA and NA subtype and showed complete correlation with conventional typing methods. The assays were highly specific and did not amplify PCR products from 3 common swine pathogens or influenza viruses other than reference strains; and have a sensitivity of detecting RNA extracted from up to 10^2 EID50 of reference virus (with original infectivity titer of 10^7 EID50/0.1 mL). Test against 40 positive samples showed that swine influenza virus H1N1, H1N2 and H3N2 were identified in 16, 1 and 20 samples, respectively, while the remaining 3 samples were detected positive for both H1N1 and H3N2 viruses. These results were 100% in agreement with those of virus isolated in embryonated eggs. Therefore the multiplex RT-PCR assays could be an effective tool for rapid detection and simultaneous subtyping of clinical swine influenza virus.