实时荧光PCR方法在A型流感病毒检测中的应用

Application of Fluorescent Real-Time Polymerase Chain Reaction in Detecting Influenza A Virus

目的比较流感病毒实时荧光PCR检测(FQ-PCR)方法与常规细胞培养、血凝试验及分型鉴定在流感病毒鉴定中的优劣,以期建立能快速简便、特异地从临床标本中检测并鉴定流感病毒的新方法。方法对76株不同型别的流感病毒分离株进行鉴定,比较两种检测方法的敏感性和特异性;对2倍梯度稀释的A型流感病毒(H1N1亚型、H3N2亚型,各1株)用FQ-PCR方法进行灵敏度检测,同时用常规细胞培养法进行病毒增殖,用血凝试验判断病毒滴度。另外,对临床采集的684份咽拭子样本直接检测,并同时进行常规培养、血凝试验及Quidel试剂检测,综合比较两种监测方法之间的差异。结果在76株流感病毒阳性细胞培养液中,FQ-PCR方法检出A型60份,同分型结果相吻合。FQ-PCR方法的灵敏度约高出常规培养法的25～26倍,无非特异性反应。结论流感病毒FQ-PCR方法具有简便快捷、特异、敏感的特点。

Objective To compare the advantage and disadvantage of fluorescent real-time polymerase chain reaction （FQ-PCR） with the conventional tissue culture method, hemagglutination test and serological subtyping influenza A virus. The aim is to establish a convenient and specific method for the identification of influenza A virus from clinical samples. Method 76 influenza virus isolates were used in the testing of the sensitivity and specificity of FQ-PCR. Two-fold serially diluted influenza A （H1N1 and H3N2 subtypes） was used in the testing of the sensitivity of FQ-PCR. The concentration of virus was determined hy hemagglutination test and titration on tissue culture. The sensitivity and specificity of FQ-PCR were also tested on 684 pharyngeal swab samples. Result 60 out of 76 infleunza virus infected cell culture supernatant were positively identified by FQ-PCR. The result was consistent with the conventional tissue culture, hemagglutination test and serological subtyping methods. The sensitivity of FQ-PCR is higher,in an order of 2^5-2^6, than the conventional tissue culture method. Conclusion FQ-PCR is a rapid, specific, and sensitive method for the detection of influenza A virus.