Development of a real-time reverse transcriptase PCR assay for type A influenza virus and the avian H5 and H7 hemagglutinin subtypes
Development of a real-time reverse transcriptase PCR assay for type A influenza virus and the avian H5 and H7 hemagglutinin subtypes
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DOI：
10.1128/jcm.40.9.3256-3260.2002
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发表时间：
2002-09-01
影响因子：
9.4
通讯作者：
Suarez, DL
中科院分区：
文献类型：
--
作者：
Spackman, E;Senne, DA;Suarez, DL
建立了一种基于禽流感病毒基质基因的实时荧光逆转录聚合酶链反应（RRT-PCR）快速检测A型流感病毒的方法。此外，基于北美禽流感病毒序列开发了H5和H7血凝素亚型特异性探针组。RRT-PCR检测采用一步RT-PCR方案和荧光水解型探针。基质基因RRT-PCR检测的检测限为10 fg或约1,000个拷贝的靶RNA，并可检测0.1 50%鸡蛋感染剂量的病毒。H5和H7特异性探针组各自具有100 fg靶RNA或约10（3）至10（4）个基因拷贝的检测限。将实时PCR检测的灵敏度和特异性与现行流感病毒检测标准进行直接比较：鸡胚中的病毒分离（VI）和血凝抑制（HI）试验的血凝素亚型分型。对1,550份来自各种鸟类的气管和泄殖腔拭子以及从纽约和新泽西活禽市场获得的环境拭子进行了比较。流感病毒特异性RRT-PCR结果与89%样本的VI结果相关。其余样品仅用一种检测方法呈阳性。总体而言，H7和H5特异性RRT-PCR的灵敏度和特异性与VI和HI相似。
A real-time reverse transcriptase PCR (RRT-PCR) assay based on the avian influenza virus matrix gene was developed for the rapid detection of type A influenza virus. Additionally, H5 and H7 hemagglutinin subtype-specific probe sets were developed based on North American avian influenza virus sequences. The RRT-PCR assay utilizes a one-step RT-PCR protocol and fluorogenic hydrolysis type probes. The matrix gene RRT-PCR assay has a detection limit of 10 fg or approximately 1,000 copies of target RNA and can detect 0.1 50% egg infective dose of virus. The H5- and H7-specific probe sets each have a detection limit of 100 fg of target RNA or approximately 10(3) to 10(4) gene copies. The sensitivity and specificity of the real-time PCR assay were directly compared with those of the current standard for detection of influenza virus: virus isolation (VI) in embryonated chicken eggs and hemagglutinin subtyping by hemagglutination inhibition (HI) assay. The comparison was performed with 1,550 tracheal and cloacal swabs from various avian species and environmental swabs obtained from live-bird markets in New York and New Jersey. Influenza virus-specific RRT-PCR results correlated with VI results for 89% of the samples. The remaining samples were positive with only one detection method. Overall the sensitivity and specificity of the H7- and H5-specific RRT-PCR were similar to those of VI and HI.