荧光定量逆转录-聚合酶链反应方法快速检测甲1型流行性感冒病毒核酸

目的建立特异、快速、敏感的TaqMan荧光定量逆转录-聚合酶链反应(RT-PCR)方法,用于检测甲1型流行性感冒(流感)病毒核酸.方法在甲1型流感病毒血凝素基因的保守区设计引物和TaqMan探针,对其进行筛选,同时对荧光定量RT-PCR反应条件和反应体系进行优化,验证该方法的特异性、敏感性、重复性,并对疑似流感患者含漱液标本进行检测.结果该方法对甲1型流感病毒的检测具有高度的特异性,对甲3型流感病毒、乙型流感病毒、禽流感病毒H5、严重急性呼吸道综合征病毒、麻疹病毒及其它呼吸道病毒均无交叉反应,检测的灵敏度达0.1TCID50.采用该方法从临床疑似患者含漱液中对甲1型流感病毒核酸的检出,比采用狗肾传代细胞进行病毒分离更为敏感.从病毒核酸提取至检测完成仅需3h左右,操作简便,重复性好,且大大减少了常规RT-PCR检测产物的污染机会.结论新建立的TaqMan荧光定量RT-PCR是一种快速检测甲1型流感病毒的特异、敏感的方法,非常适合于疑似流感爆发疫情的应急诊断和鉴别诊断.

TaqMan-based Real-time RT-PCR Assay for Quick Detection of Influenza A1 Virus RNA

Objective To establish a TaqMan-based real-time PCR assay for detection of influenza A_1 virus RNA.Methods The specific primers and probes were designed in the conserved region of influenza A_1 virus the HA gene.The primers and probes are selected,the reactive condition was and system optimized to improve the sensitivity,specificity and repetition of the assay.The clinical specimens collected from the patients with acute respiratory tract infection were detected by this assay.Results The specificity of the assay was high and there were no cross reactions with influenza A_3,B,A_5 virus SARS virus,measles virus and the other common respiratory virus.The sensitivity of the assay was 0.1 TCID_(50);Specificity and sensitivity of the rea-time PCR method were compared to the conventional virus isolating using MDCK cells the detection rate of influenza A_1 virus from the throat of the clinical patients,washing specimens by RT-PCR method was much higher than that by conventional method.It took only three hours to extract viral RNA and do the real-time PCR.It had advantages of contamination control,automation and direct virus quantification.Conclusion This TaqMan-based real-time PCR assay was a quick,sensitive and specific tool for molecular diagnosis of influenza A_1 virus.