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目的 :建立快速、敏感、特异的虫媒病毒RT_PCR检测方法。方法 :采用改进的核酸制备方法 ,分别从感染的乳鼠脑和细胞培养上清液中提取病毒RNA ,再用RT_PCR和套式PCR方法进行检测。结果 :通过对RT_PCR反应条件的优化 ,用 3种虫媒病毒的特异引物均可从不同稀释度的感染小鼠脑和细胞上清中提取的病毒RNA扩增出特异的目的基因片段。甲病毒属的东部马脑炎病毒和西部马脑炎病毒之间 ,及与黄病毒属的黄热病毒均无交叉反应 ,表明建立的RT_PCR检测方法特异性强。套式PCR与RT_PCR比较可提高检测敏感性 10～ 10 0 0 0倍。结论 :建立的RT_PCR和套式PCR法可用于 3种虫媒病毒的快速检测。
Objective: To establish a rapid, sensitive and specific RT_PCR detection method for arbovirus. Methods: The viral RNA was extracted from the infected neonatal rat brain and cell culture supernatant by using the improved method of nucleic acid preparation. RT - PCR and nested PCR were used to detect the viral RNA. Results: By optimizing the reaction conditions of RT_PCR, specific target gene fragments were amplified from virus RNA extracted from brain and cell supernatant of infected mice with different dilutions by using three kinds of special primers of arbovirus. There was no cross reaction between the alpha equine Encephalitis virus and western equine encephalitis virus and the yellow fever virus of Flaviviridae, indicating that the established RT-PCR detection method is specific. Nested PCR and RT_PCR can improve the detection sensitivity of 10 ~ 100 times. Conclusion: The established RT_PCR and nested PCR method can be used for rapid detection of three kinds of arbovirus.