荧光定量RT-PCR快速检测乙型流感病毒核酸

目的建立以特异性荧光探针为特点的TaqMan荧光定量RT-PCR方法用于检测乙型流感病毒核酸.方法根据GenBank登录的流感毒株序列,应用生物学软件进行序列比对,在乙型流感病毒核蛋白(NP)基因的保守区设计引物和TaqMan探针进行筛选,对荧光定量RT-PCR反应条件进行优化,检测该方法的特异性和灵敏度.此外还对疑似乙型流感含漱液标本进行检测.结果该方法对乙型流感病毒的检测有高度的特异性,对甲1型、甲3型、禽流感病毒H5、SARS病毒及其他呼吸道病毒均无交叉反应,检测的灵敏度达0.01 TCID50,可从疑似流感患者含漱液中直接检测流感病毒核酸,从病毒核酸提取至完成检测仅需3 h左右.结论本研究建立的TaqMan荧光定量RT-PCR是一种快速检测乙型流感病毒特异、敏感的新方法.

TaqMan-based Real-time PCR Assay for Quick Detection of Influenza B Virus

Objective To establish a TaqMan based real time PCR assay for the detection of influenza B virus.Methods The HA gene of influenza B virus downloaded from the Genbank was aligned using the biologic software and the specific primers and probes were designed in the conserved region of the NP gene. The primers, probes and the reactive condition were optimized to improve the sensitivity and specificity of the assay. The clinical specimens collected from the patients with acute respiratory tract infection were detected. Results The specificity of the assay was high and there were no cross reactions with influenza A1,A3 and A5 virus, SARS virus and other common respiratory virus. The sensitivity of the assay was 0 01TCID 50 and the viral RNA was directly detected from the clinical specimens by this assay. It took only three hours to extract viral RNA and do the real time PCR.Conclusions The TaqMan based real time PCR assay was a quick, sensitive and specific method for molecular diagnosis of influenza B virus.