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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 method for the detection and identification of influenza A (Flu A) and hemagglutinin H3, H5 and H7 genotypes by quantitative real-time RT-PCR. Methods RNase P (RNase P) gene was used as an internal reference and Primer Express 3.0 software was used to design PCR specific primers and probes. Plasmid standards were constructed to analyze the sensitivity and reproducibility of the method and specific assays were performed using respiratory virus samples from different sources. Results The sensitivities of the Flu A, H3, H5, H7 and RNase P plasmids were all 102 copies / m L and the specificity was 100%. Only the corresponding viruses were detected by each pair of primers and probes and no cross reaction , Coefficient of variation (CV) ≤1.99%. Conclusion The established five-fold fluorescence quantitative RT-PCR method has high sensitivity, specificity and repeatability, and can be used to detect a variety of subtypes of influenza A, which has certain value in the early diagnosis of different subtypes of influenza A virus.