李痘病毒实时荧光定量RT-PCR检测方法的建立

Development of a Real-Time Fluorescent Quantitative RT-PCR Method for the Detection of Plum Pox Virus

目的:建立李痘病毒(PPV)特异、灵敏、快速的实时荧光定量RT-PCR检测方法,用于核果类种苗的健康评测及李痘病毒疫情监测。方法:根据PPV-D株系和PPV-M株系的外被蛋白(CP)基因保守序列,设计特异性引物和TaqMan探针,扩增全长CP基因片段,并将其克隆到pMD18-T载体上,构建质粒标准品,建立PPV的实时荧光定量RT-PCR检测方法,并对该方法的特异性、灵敏度和重复性进行评估。结果:此荧光定量RT-PCR方法对PPV检测呈现高灵敏度和高特异性,与马铃薯Y病毒和马铃薯X病毒无交叉反应,最低检出限可达1.6×102拷贝/μL,标准曲线的相关系数为0.999 18。结论:建立了李痘病毒的荧光定量RT-PCR检测方法,可望应用于检验检疫部门对李痘病毒的快速检测。

Objective：To establish a real-time PCR method for detection and quantification of Plum pox virus（PPV）.Methods：The primers and probes were designed and synthesized according to the sequences for coat protein gene of PPV-D and PPV-M.The genes were amplified by RT-PCR and cloned to the pMD18-T vector.After the detection method of PPV was established,its specificity,sensitivity and reproducibility were estimated.Results：The results showed that the assay method developed possessed the character of high accuracy and repetition,without cross-reaction with Potato virus X（PVX） and Potato virus Y（PVY）.The sensitivity was as low as 1.6×10^2 copies/μL per reaction.The correlation coefficient of the standard curve was 0.999 18.Conclusion：It suggested that the detection method of PPV was established by real-time RT-PCR,and could be used for the rapid detection of PPV at entry-exit inspection and quarantine