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

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.