H1、H3亚型猪流感病毒双重实时荧光定量RT-PCR检测方法的建立及应用

为建立一种快速、准确地检测H1和H3亚型猪流感病毒（swine influence virus，SIV）的方法，根据H1和H3亚型SIV血凝素（hemagglutinin，HA）基因保守序列，分别设计2对特异性引物和2条TaqMan探针，建立双重实时荧光定量RT-PCR方法。结果显示，该方法敏感性高，可检测到最低拷贝数为10²拷贝/μL；重复性良好，重复孔Ct值的变异系数均在5%以下；特异性好，除H1和H3亚型SIV外，H4、H5、H7、H9亚型SIV、猪瘟病毒、猪繁殖与呼吸综合征病毒、口蹄疫病毒、伪狂犬病病毒检测均为阴性；在田间样品中成功检出1株H1和1株H3亚型SIV，检出率为1.16%。该方法准确、快速、灵敏、特异，可为H1、H3亚型SIV的快速鉴别检测及流行病学调查提供有效的技术手段。

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.