猪流感病毒H1、H3、N1、N2亚型分型 RT-PCR方法的建立

根据GenBank中H1N1和H3N2亚型猪流感病毒(SIV)血凝素(hemagglutinin,HA)、神经氨酸酶(neuraminidase,NA)和M基因保守序列,分别设计合成了5对特异性引物,利用RT-PCR技术对SIV的型和亚型进行鉴定。结果表明,该方法的型RT-PCR可以检测出10⁴ EID₅₀病毒量所提取的RNA;H1、H3、N1和N2的亚型RT-PCR均可以检测出10⁴ EID₅₀病毒量所提取的RNA。除每对特异性引物所对应的亚型外,对其他亚型及猪繁殖与呼吸综合征病毒(PRRSV)和猪瘟病毒(CSFV)的检测均为阴性,应用该方法对临床样品进行检测,其结果与病毒分离结果符合率为100%。结果表明,该方法特异性好、敏感性高,有望成为SIV的一种特异、敏感、快速的分型检测方法,为猪流感分子流行病学的调查奠定了良好的基础。

Development of RT-PCR Assay for Rapid Detection and Subtyping of Swine Influenza Virus H1, H3, N1, N2

According to the genomic sequence of H1N1,H3N2 subtype swine influenza virus(SIV) published in GenBank,the specific primers were designed according to the conserved region of H1,H3 HA,N1,N2 NA and M genes of swine influenza virus(SIV) H1N1,H3N2 subtype.RT-PCR assay was developed for detection of swine influenza H1,H3,N1,N2 subtype.The results showed that the RT-PCR was capable of detecting 104 EID50 of M and 104 EID50 of HA or NA.The results were negative for the detection of porcine reproductive and respiratory syndrome virus,CSFV and other subtypes except the corresponding subtype.The coincidence rates between RT-PCR and virus isolation were 100%.This assay provides a rapid,sensitive,and cost-effective laboratory diagnosis for detecting and subtyping of SIV in pigs.