人类流感病毒多重PCR分型方法的建立  

Multiplex RT-PCR for typing and subtyping of human influenza viruses

作　　者：林方[1] 熊伟[1] 康晓平[1] 李永强[1] 刘洪[1] 李辉[2] 祝庆余[1] 杨银辉[1] 

机构地区：[1]军事医学科学院微生物流行病研究所病原微生物生物安全国家重点实验室 [2]71781部队卫生队

出　　处：《军事医学科学院院刊》2010年第4期331-333,344,共4页Bulletin of the Academy of Military Medical Sciences

基　　金：国家科技重大专项(2008ZX10004-001-13)

摘　　要：目的建立一种可以同时检测人类A型流感病毒,B型流感病毒及A型流感病毒H1,H3,H5亚型的多重RT-PCR方法。方法根据人类流感病毒的特异性基因设计并合成多对特异性引物,建立可同时扩增出人类流感病毒亚型特异性片段的多重PCR的方法。同时,对该方法的特异性和敏感性进行了评价,并利用该方法对39份疑似swineH1N1的临床标本进行检测。结果该方法可同时扩增出A型流感病毒M基因203bp,B型流感病毒M基因296bp,A型流感病毒亚型的HA基因片段长度分别为362bp(H1),112bp(H3),188bp(H5),494bp(swineH1)。该实验最低可检测新甲型H1N1(A/Beijing/501/2009)病毒RNA0.5TCID50。该方法的特异性实验结果显示,在多重PCR检测中未检出非特异扩增的PCR产物。同时,使用该方法从39份疑似新甲型H1N1的临床咽拭子标本中检测出新甲型H1N1阳性的标本4份(10.2%),季节性H1N1亚型2份(5.1%),H3N2亚型12份30.7%,本次实验结果与WHO通过实时PCR方法检测的结果一致。结论本研究建立的多重PT-PCR方法快速、敏感,特异性强,可用于人类流感病毒的分型检测。Objective To develop a multiplex reverse transcription-polymerase chain reaction （RT-PCR） to simultaneously type human influenza A virus and influenza B virus,and to subtype H1,H3,H5 and novel H1（swine H1） of human influenza virus type A.Method Primers for typing influenza viruses were targeted to conserved regions and a multiplex RT-PCR for typing and subtyping human influenza viruses was established.Results The type-specific primers were designed to amplify MA fragments of 214 bp for influenza A virus,and 296 bp for influenza B virus.The subtype-specific primers were designed to amplify HA fragments of 362 bp for H1,112 bp for H3,188 bp for H5 and 494 bp for novel H1,respectively.The sensitivity of the multiplex RT-PCR was evaluated by testing the novel H1N1 strain （A/Beijing/501/2009） and the synthetic full-length H1 gene of novel H1N1 virus（A/California/04/2009）.The testing limit was 0.5 TCID50/reaction for the novel H1N1 virus.Clinical throat swab specimens collected from 39 suspected cases of novel A H1N1 patients were tested to validate the multiplex assay.Four of the specimens （10.2%） were confirmed as novel A （H1N1） positive,two were identified as seasonal H1N1 （5.1%） and 12 cases as H3N2 （30.7%）,which were identical to real-time PCR assay recommended by WHO.Conclusion The developed multiplex RT-PCR assay is rapid,sensitive and specific for typing and subtyping human influenza viruses.

关 键 词：人类流感病毒 病毒分型 多重PCR 

分 类 号：R440[医药卫生—诊断学]