普马拉病毒实时荧光定量RT-PCR方法的建立

　　杨鹏飞1,2，燕清丽1,2，张丽萍1，徐焕洲1，王玉春1，甄维1，胡孔新1*

　　1.中国检验检疫科学研究院卫生检验检疫研究所，北京 100123；2.淮安市疾病预防控制中心

　　摘要：目的 建立一种适应国境口岸地区特定鼠种中普马拉病毒实时荧光定量RT-PCR检测方法。方法 利用BeaconDesigner7.0软件设计引物和探针，以人工合成普马拉病毒S基因的片段作为模板，进行实时荧光定量RT-PCR检测，并验证该方法的灵敏度及特异性。结果模板的Ct值与模板稀释浓度的对数存在良好的线性关系，标准曲线y = -3.122 x + 38.605，R2　=　0.995，PCR扩增效率为109.1%，其最低检出限为31.6 copies/μl。结论建立的实时荧光定量RT-PCR方法特异性好、灵敏度高，适合于普马拉病毒的快速检测。

　　关键词：普马拉病毒;定量;实时荧光RT-PCR

　　中图分类号：R373 文献标识码：B

　　Establishment of quantitative real-time RT-PCR of Puumalavirus

　　YANG Peng-fei*, YAN Qing-li, ZHANG Li-ping, XU Huan-zhou, WANGYu-chun, ZHENG Wei, HU Kong-xin

　　Chinese Academy of Inspection and Quarantine, Beijing100123,China

　　Abstract:   Objective   To establish aquantitative real-time fluorescence RT-PCR (qRT-PCR) method fordetecting Puumala virus(PUUV) from some rodents at frontierports.   Methods    The twin primers andthe TaqMan probe were designed and synthesized based on the S genesegment of PUUV, and the real-time RT-PCR was developed for testingthe sensitivity and the specialty.   Results  The Ct value of templates had a linear relationship with the logstarting quantity. Sensitivity assay showed that the detectionlimit of the assay was 31.6 copies/μl and the standard curve y =-3.122 x + 38.605 had a good reproducibility. Conclusion   Quantitative real-time fluorescence for PUUVdetection was established and characterized by rapidity andsensitivity.

　　Key words:   Puumala virus; Quantitative; Real-timeRT-PCR

　　《中国国境卫生检疫杂志》2013年8月刊