3种重要虫媒病毒的RT-PCR检测

Detection of three important arbovirus strains by reverse transcription polymerase chain reaction

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 reverse transcription polymerase chain reaction (RT_PCR) method for rapid, sensitive, and specific detection of arbovirus.Methods:Genomic viral RNAs were extracted from infected suckling mice brain and culture cell supernatant by a modified method of nucleic acid preparation. Then the viral RNA was detected by RT_PCR and nested PCR.Results:Specific DNA fragments could be amplified with the optimized conditions from extracted RNAs of all four arbovirus species. No cross reactions were observed between eastern equine encephalitis virus,western equine encephalitis and yellow fever virus strains, which suggested a high specificity of established RT_PCR method. Nested PCR could increase the sensitivity by 10- 10?000 folds compared with RT_PCR.Conclusion:RT_PCR and nested PCR established in the study could be applied in rapid detection of three arbovirus strains. [