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首页> 外文期刊>Journal of Clinical Microbiology >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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Development of a Real-Time Reverse Transcriptase PCR Assay for Type A Influenza Virus and the Avian H5 and H7 Hemagglutinin Subtypes

机译：甲型流感病毒以及禽类H5和H7血凝素亚型实时逆转录酶PCR检测方法的开发

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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 103 to 104 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.
机译：开发了一种基于禽流感病毒基质基因的实时逆转录PCR（RRT-PCR）方法，用于快速检测A型流感病毒。此外，H5和H7血凝素亚型特异性探针组是根据北美禽流感病毒序列开发的。 RRT-PCR分析利用一步式RT-PCR方案和荧光水解型探针。基质基因RRT-PCR分析的检测极限为10 fg或大约1,000拷贝的目标RNA，并且可以检测到0.1 50％的鸡蛋感染剂量的病毒。 H5和H7特异性探针组的检测极限分别为100 fg靶RNA或大约10 3 至10 4 基因拷贝。将实时PCR检测的灵敏度和特异性直接与当前用于检测流感病毒的标准检测的灵敏度和特异性进行了比较：胚胎鸡蛋中的病毒分离（VI）和通过血凝抑制（HI）检测的血凝素亚型分析。比较了1,550种来自各种鸟类的气管和泄殖腔拭子，以及从纽约和新泽西州的活禽市场获得的环境拭子。流感病毒特异性RRT-PCR结果与89％样品的VI结果相关。其余样品仅用一种检测方法为阳性。总体而言，H7和H5特异性RRT-PCR的敏感性和特异性与VI和HI相似。

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