H3N2亚型禽流感病毒双重实时荧光定量RT-PCR检测方法的建立

Development of a Duplex Real-time PCR for Detection of H3N2 Avian Influenza Virus

为建立快速、简便检测H3N2亚型禽流感病毒(AIV)的方法,根据H3和N2亚型AIV HA、NA基因保守序列,分别设计筛选了特异性引物和用不同荧光基团标记的TaqMan探针。通过优化反应条件,建立了H3N2亚型AIV双重实时荧光定量RT-PCR检测方法。结果显示该法特异性强,不与其他亚型AIV和常见禽病病原体发生交叉反应,敏感性达100拷贝/μL;组内组间重复性好,平均变异系数均小于3%;应用该法对96份临床样品检测,结果与病毒分离鉴定结果一致。表明本研究建立的H3N2亚型AIV双重实时荧光定量RT-PCR具有快速、特异、敏感、重复性好等优点,可用于H3N2亚型AIV的快速诊断和监测。

In order to establish a quick and simple method to detect H3N2 subtype avian influenza virus （AIV） ,the specific primers and TaqMan probes were designed according to the conserved sequences of the hemagglutinin （HA） genes and the neuramidinase （NA） genes of H3 and N2 subtype AIV,respectively. The reaction conditions were optimized and the duplex real-time PCR assay for rapid detection of H3N2 subtype AIV was established. The results showed that the specificity of this assay was high and the detec- tion limit of this assay was 100 copies/μL of H3N2 AIV;The coefficients of variation were both less than 3% for the intra-assay and inter-assay; The detection of clinical samples were accorded with the viral isola- tion completely. This duplex real-time PCR assay is a quick, sensitive and specific method for the detection of H3N2 subtype AIV and will be useful for the rapid diagnosis and monitoring of H3N2 AIV.