李痘病毒实时荧光定量RT-PCR检测方法的建立

摘要

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 iow as 1.6×102 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.