Development and Validation of RT-PCR Tests for the Detection and S1 Genotyping of Infectious Bronchitis Virus and Other Closely Related Gammacoronaviruses Within Clinical Samples
Development and Validation of RT-PCR Tests for the Detection and S1 Genotyping of Infectious Bronchitis Virus and Other Closely Related Gammacoronaviruses Within Clinical Samples
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DOI：
10.1111/j.1865-1682.2011.01222.x
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发表时间：
2011-10-01
影响因子：
4.3
通讯作者：
Wakeley, P. R.
中科院分区：
文献类型：
--
作者：
Jones, R. M.;Ellis, R. J.;Wakeley, P. R.
开发了两种检测方法，可以对传染性支气管炎病毒（IBV）和其他密切相关的γ冠状病毒进行检测和基因分型。第一种测试采用一步逆转录聚合酶链反应（RT-PCR）测定，其中使用TaqMan（R）探针在真实的时间内监测扩增。该实时RT-PCR测试用于检查一组现场样品，并将其性能与含胚禽蛋中的病毒分离进行比较。共检测了323份现场样品; 176份样品采用实时RT-PCR方法检测呈阳性，但只有3份样品通过病毒分离呈阳性。测序用于确认样本子集的阳性实时RT-PCR结果。该测试适用于拭子和尸检样本，并已被证明具有高度灵敏度和特异性。第二种检测方法是基因分型方法，用于根据编码表面刺突（S）糖蛋白S1亚基的基因内的核苷酸变异鉴定田间样品中存在的IBV毒株。开发该方法是为了提供一种工具，为疫苗接种决策提供信息，并进行持续监测，以检测英国境内新出现的IBV毒株。使用IBV的实验室分离株和现场样品评价了试验的性能。这两种检测方法都适用于高通量诊断实验室。
Two tests were developed that allow the detection and genotyping of infectious bronchitis virus (IBV) and other closely related gammacoronaviruses. The first test employs a one-step, reverse transcription-polymerase chain reaction (RT-PCR) assay in which the amplification is monitored in real time using a TaqMan (R) probe. This real-time RT-PCR test was used to examine a panel of field samples and its performance compared to virus isolation in embryonated fowls' eggs. A total of 323 field samples were tested; 176 samples were positive using the real-time RT-PCR method, but only three were positive by virus isolation. Sequencing was used to confirm the positive real-time RT-PCR results for a subset of samples. The test is suitable for swabs and post-mortem samples and has been shown to be highly sensitive and specific. The second test, a genotyping method, was developed for identification of the strain of IBV present in field samples based on nucleotide variations within the gene encoding the S1 subunit of the surface spike (S) glycoprotein. This method was developed to provide a tool to inform vaccination decisions and for ongoing surveillance to detect new and emerging strains of IBV within the UK. The performance of the test was evaluated using laboratory isolates of IBV and field samples. Both tests are suitable for use in a high-throughput diagnostic laboratory.