Taq Man荧光定量RT-PCR快速检测甲3型流感病毒

Rapid detection of influenza A3 virus by TaqMan-based real-time RT-PCR assay

目的建立一种特异、灵敏、快速的荧光定量RTPCR方法用于检测甲3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,在甲3型流感病毒血凝素(HA)基因的保守区设计引物和TaqMan探针、并进行筛选。对荧光RT-PCR反应条件进行优化,检测该方法的特异性和灵敏度。并对疑似流感含漱液标本进行检测。结果该方法对甲3型流感病毒的检测有高度的特异性,对甲1型、乙型、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0.01TCID50,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3h左右。结论本研究建立的TaqMan荧光定量RTPCR是一种快速检测甲3型流感病毒特异、敏感的新方法。

To establish a specific, sensitive method of TaqMan-based real time RT-PCR assay for the rapid detection of influenza A3 virus, the hemagglutinin (HA) gene of influenza virus down-loaded from Genbank was aligned by using the biologic software, and the specific primers and probes were designed in the conserved regions of HA gene. The primers and probes as well as the reaction condition were optimized to improve the sensitivity and specificity of the assay. The throat swab specimens used in this assay were taken from patients with acute respiratory tract infections. It was found that the specificity of this assay was high without any cross-reactions with influenza A1, A5 ,B viruses, SARS virus and other commonly encountered viruses. The sensitivity of this assay was 0.01 TCID_~50 , and the viral RNA could be detected directly from the clinical specimens. It took only 3 hours to complete the whole course of reaction including extraction of viral RNA and the real-time PCR. It concludes that the TaqMan-based real-time RT-PCR assay is a rapid, sensitive and specific method for the detection of influenza A3 virus.