普马拉病毒实时荧光定量RT-PCR方法的建立  

Establishment of quantitative real-time RT-PCR of Puumala virus

作　　者：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] 

机构地区：[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

出　　处：《中国国境卫生检疫杂志》2013年第4期220-222,227,共4页Chinese Journal of Frontier Health and Quarantine

基　　金：国家质检总局科技计划项目(2011IK150);中国检科院科研项目(2011JK008)

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Beacon Designer7.0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=-3.122x+38.605,R2=0.995,PCR扩增效率为109.1%,其最低检出限为31.6copies/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。Objective To establish a quantitative real-time fluorescence RT-PCR （qRT-PCR） method for detecting Puumala virus（PUUV） from some rodents at frontier ports. Methods The twin primers and the TaqMan probe were designed and synthesized based on the S gene segment of PUUV, and the real-time RT-PCR was developed for testing the sensitivity and the specialty. Results The Ct value of templates had a linear relationship with the log starting quantity. Sensitivity assay showed that the detection limit 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 PUUV detection was established and characterized by rapidity and sensitivity.

关 键 词：普马拉病毒 定量 实时荧光RT-PCR 

分 类 号：R373[医药卫生—病原生物学]