禽流感H5、H7、H9亚型多重实时荧光RT-PCR检测方法的建立  

Development of a Multiplex Real-time Reverse Transcriptase PCR Assay for Simultaneous Identification of Avian H5,H7 and H9 Hemagglutinin Subtypes

作　　者：秦智锋[1] 吕建强[1] 肖性龙 钟安清[1] 林镜中 黄运生[1] 林庆燕[1] 陈书琨[1] 金显忠[1] 林苗[1] 刘胜利[1] 

机构地区：[1]深圳出入境检验检疫局,深圳518045 [2]深圳太太基因工程有限公司,深圳518057

出　　处：《病毒学报》2006年第2期131-136,共6页Chinese Journal of Virology

基　　金：本课题为深圳出入境检验检疫局科学研究与技术开发重大项目(SZK01-2004)

摘　　要：为了对致病性强、危害性大的H5、H7、H9亚型禽流感病毒进行同时集成化快速检测,通过对GenBank已报道的禽流感病毒的HA基因进行序列分析比较,设计了H5、H7、H9 3个亚型的特异性引物和分别用3个荧光基团标记的Taqman MGB核酸探针。将各个亚型引物与探针优化组合,筛选出能够同时检测禽流感病毒H5、H7、H9 3个亚型、且对Ct值和扩增效率影响不大的3组引物和探针,建立了三重实时荧光RT-PCR方法。该方法特异性好,在我们检测的样品中,没有发现假阳性和假阴性现象。同时敏感性高,检测禽流感病毒H5、H7、H9亚型的敏感性分别达到1 0001、000、500个模板拷贝数;此外抗干扰能力强,对禽流感H5、H7、H9 3个亚型的不同模板浓度进行组合,仍可有效地同时检测3个病毒亚型。所建立的方法对保存的89个禽流感病毒样品进行检测,结果与经典检测方法(病毒分离鉴定、HA、HI)的符合率达100%。用上述建立的方法与鸡胚分离法同时对新鲜采集的4 000多份临床样品进行检测,两种方法的检测结果符合率为100%。In order to simultaneously and massively identify the avian influenza virus subtypes of H5,H7 and H9 which are pathogenic and dangerous, HS, H7 and H9 hemagglutinin subtype-specific Taqman MGB probe marked with three different primer sets were developed based on all avian influenza virus HA sequences published in GenBank. A multiplex real-time reverse transcriptase PCR （RRT-PCR） assay was developed for the rapid detection of subtypes H5, H7 and H9 of avian influenza virus. The multiplex RRT-PCR assay was specific and no false positive or false negative results were found. The sensitivity of multiplex RRT-PCR assay was 1000,1000,500 template copies for H5, H7 and H9 respectively. Different concentrations of H5, H7 and H9 when mixed together still could be identified by this assay, which implied the assay could be applied to clinical confirmation for simultaneous infection of AIVs. 89 AIV strains that preserved in Shenzhen Entry-Exit Inspection and Quarantine Bureau were all reidentified by this assay, and the results were consistent with those of currently used methods, virus isolation in embryonated chicken eggs and hemagglutinin subtyping by hemagglutination inhibition （HI） assay. A comparison was performed with 4000 fresh tracheal and cloacal swab specimens from various avian species obtained from live-bird farms in Shenzhen. Influenza virus-specific RRT-PCR results of the samples were 100% correlated with virus isolation result.

关 键 词：禽流感 H5亚型 H7亚型 H9亚型 实时荧光RT PCR 多重实时检测 

分 类 号：S852.65[农业科学—基础兽医学]