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首页> 美国卫生研究院文献>PLoS Clinical Trials >Surveillance for Western Equine Encephalitis, St. Louis Encephalitis, and West Nile Viruses Using Reverse Transcription Loop-Mediated Isothermal Amplification
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Surveillance for Western Equine Encephalitis, St. Louis Encephalitis, and West Nile Viruses Using Reverse Transcription Loop-Mediated Isothermal Amplification

机译：使用逆转录环介导的等温扩增对西方马脑炎，圣路易斯脑炎和西尼罗河病毒进行监测

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摘要

Collection of mosquitoes and testing for vector-borne viruses is a key surveillance activity that directly influences the vector control efforts of public health agencies, including determining when and where to apply insecticides. Vector control districts in California routinely monitor for three human pathogenic viruses including West Nile virus (WNV), Western equine encephalitis virus (WEEV), and St. Louis encephalitis virus (SLEV). Reverse transcription quantitative polymerase chain reaction (RT-qPCR) offers highly sensitive and specific detection of these three viruses in a single multiplex reaction, but this technique requires costly, specialized equipment that is generally only available in centralized public health laboratories. We report the use of reverse transcription loop-mediated isothermal amplification (RT-LAMP) to detect WNV, WEEV, and SLEV RNA extracted from pooled mosquito samples collected in California, including novel primer sets for specific detection of WEEV and SLEV, targeting the nonstructural protein 4 (nsP4) gene of WEEV and the 3’ untranslated region (3’-UTR) of SLEV. Our WEEV and SLEV RT-LAMP primers allowed detection of <0.1 PFU/reaction of their respective targets in <30 minutes, and exhibited high specificity without cross reactivity when tested against a panel of alphaviruses and flaviviruses. Furthermore, the SLEV primers do not cross-react with WNV, despite both viruses being closely related members of the Japanese encephalitis virus complex. The SLEV and WEEV primers can also be combined in a single RT-LAMP reaction, with discrimination between amplicons by melt curve analysis. Although RT-qPCR is approximately one order of magnitude more sensitive than RT-LAMP for all three targets, the RT-LAMP technique is less instrumentally intensive than RT-qPCR and provides a more cost-effective method of vector-borne virus surveillance.
机译：收集蚊子和检测媒介传播的病毒是一项关键的监视活动，它直接影响公共卫生机构的媒介控制工作，包括确定何时何地使用杀虫剂。加利福尼亚的病媒控制区定期监视三种人类致病病毒，包括西尼罗河病毒（WNV），西马脑炎病毒（WEEV）和圣路易斯脑炎病毒（SLEV）。逆转录定量聚合酶链反应（RT-qPCR）可在单个多重反应中对这三种病毒进行高度灵敏和特异的检测，但是该技术需要昂贵的专用设备，通常只能在中央公共卫生实验室中使用。我们报告了使用逆转录环介导的等温扩增（RT-LAMP）来检测从加利福尼亚州收集的合并蚊子样本中提取的WNV，WEEV和SLEV RNA，包括针对非结构性WEEV和SLEV的特异性检测的新型引物组WEEV的蛋白4（nsP4）基因和SLEV的3'非翻译区（3'-UTR）。我们的WEEV和SLEV RT-LAMP引物允许在<30分钟内检测到<0.1 PFU /各自靶标的反应，并且在针对一组alpha病毒和黄病毒进行测试时表现出高特异性而没有交叉反应。此外，尽管两种病毒都是日本脑炎病毒复合体的密切相关成员，但SLEV引物不会与WNV发生交叉反应。 SLEV和WEEV引物也可以在单个RT-LAMP反应中结合，通过熔解曲线分析区分扩增子。尽管对于所有三个靶标，RT-qPCR的灵敏度都比RT-LAMP高大约一个数量级，但RT-LAMP技术的仪器强度不如RT-qPCR，并且提供了一种更具成本效益的载体传播病毒监测方法。

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