四重荧光定量RT-PCR检测人感染新型甲型禽流感H7N9病毒

A four multiplex fluorescent quantitative RT-PCR assay for the detection of novel avian-origin influenza A (H7N9) virus in human infections

目的建立检测人感染新型甲型禽流感H7N9病毒的四重荧光定量RT-PCR方法。方法以人细胞RNA酶P(RNase P)基因为内参,用Primer Express3.0软件设计PCR特异性引物和探针。通过不同来源的呼吸道病毒进行特异性分析,构建质粒标准品用于分析该方法的灵敏度和重复性,并对1 896例临床标本进行回顾性检测。结果该方法检测甲型流感病毒(Flu A)、H7、N9与RNase P灵敏度均达102copies/m L,特异性达100%,每对引物和探针只检测出相应的病毒,无交叉反应,检测变异系数(CV)均低于1.55%。用该法检测1 896例临床标本,结果 Flu A 235例,H7N9 127例,与其他试剂报告结果完全一致。结论建立的四重荧光定量RT-PCR法可快速检测H7N9病毒,灵敏度与特异性好,可用于H7N9病毒感染患者的早期诊断。

Objective To establish a four multiplex fluorescent quantitative ＇RT-PCR assay for the detection of novel avian-origin influ- enza A（H7N9） virus in human infections. Methods The primers and probes for H7N9 virus and RNase P gene（internal control） were designed with Primer Express 3.0 software. The specificity of the multiplex RT-PCR assay was determined by testing different kinds of respirovirus, and its sensitivity and repeatability by the constructed standard plasmid. A total of 1 896 clinical specimens were retrospectively detected by the established method. Results The analytical sensitivities of the established multiplex RT-PCR assay for Flu A, H7, N9 and RNase P reached to 102 copies/mL, and the specificity 100%. No cross-reaction was observed, and each pair of primers and probes seriously corresponded to the detection of each virus. The coefficients of variation（CV） were all less than 1.55%. Of the 1 896 clinical specimens, 235 were positive in Flu A and 127 positive in HTN9, which were consistent with the results deter- mined by other methods. Conclusion The established four multiplex fluorescent quantitative RT-PCR assay with good sensitivity and specificity can detect H7N9 virus rapidly, which may be suited to the early diagnosis of the patients with H7N9 virus infection. Key words： avian-origin influenza A; H7Ng; detection; RNase P; four multiplex fluorescent quantitative RT-PCR