五重荧光定量RT-PCR法检测甲型流感病毒

Detection of influenza A virus by multiplex real-time RT-PCR assay

目的建立一种五重荧光定量RT-PCR检测并鉴别流感病毒A(Flu A)及血凝素H3、H5、H7基因亚型的方法。方法以人细胞核糖核酸酶P(RNase P)基因为内参照,用Primer Express 3.0软件设计PCR特异性引物和探针。构建质粒标准品用于分析该方法的灵敏度和重复性,利用不同来源的呼吸道病毒样本进行特异性分析。结果该法检测Flu A、H3、H5、H7以及RNase P质粒标准品的灵敏度均达102copies/m L,特异性达100%,各对引物和探针仅检测出相应的病毒,未有交叉反应,变异系数(CV)≤1.99%。结论建立的五重荧光定量RT-PCR法灵敏度、特异性高,重复性好,可用于检测出多种甲型流感亚型,对甲型流感病毒不同亚型早期诊断具有一定的价值。

Objective To establish a multiplex real-time RT-PCR assay to detect and discriminate influenza A virus（ Flu A）,hemagglutinin 3（ H3）,hemagglutinin 5（ H5） and hemagglutinin 7（ H7） in a single test tube. Methods Using the Primer Express 3. 0 software,the primers and probes of PCR for the above-mentioned viruses were designed. The primer and probe set specifically targeted to the human nucleic acid RNase P（ RP） which was used as the internal control were also designed. The analytical sensitivity was determined by serial dilutions of each virus,and the specificity of multiplex assay was determined by tests for different virus. Results The analytical sensitivities of the multiplex real-time RT-PCR assay for Flu A,H3,H5,H7 and RNase P reached to 102 copies / m L and the specificity was 100%. No cross-reaction was observed for the mixed viruses and common respiratory pathogens and commensal organisms. The coefficients of variation（ CV） were all less than 1. 99%. Flu A,H3,H5,H7 and RNase P were successfully detected by the established multiplex assay. Conclusion A sensitive and specific multiplex real-time RT-PCR assay was developed to detect Flu A,H3,H5,H7 and RNase P. The multiplex RT-PCR should be valuable for clinical diagnosis of the different subtype of influenza A virus.