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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 real-time fluorescence quantitative RT-PCR method for detecting Pumala virus in specific mouse species at border crossings. Methods Primers and probes were designed with Beacon Designer7.0 software. The fragment of S gene of Pumala virus was synthesized and used as a template for real-time fluorescence quantitative RT-PCR. The sensitivity and specificity of this method were also verified. Results There was a good linear relationship between the Ct value of the template and the logarithm of the dilution of the template. The standard curve y = -3.122x + 38.605, R2 = 0.995, PCR amplification efficiency was 109.1%, and the minimum detection limit was 31.6copies / μl . Conclusion The established real-time fluorescence quantitative RT-PCR method has good specificity and high sensitivity, and is suitable for the rapid detection of Pumala virus.