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李痘病毒实时荧光定量RT-PCR检测方法的建立
引用本文:高雅红,王进忠,李明福,李桂芬.李痘病毒实时荧光定量RT-PCR检测方法的建立[J].生物技术通讯,2011,22(1):77-80.
作者姓名:高雅红  王进忠  李明福  李桂芬
作者单位:1. 北京农学院,北京,102206;中国检验检疫科学研究院,北京,100029
2. 北京农学院,北京,102206
3. 中国检验检疫科学研究院,北京,100029
基金项目:国家自然科学基金(30971938); 国家“十一五”科技支撑计划(2006BAD08A13,2006BAD08A16-1); 北京市教育委员会项目(KM200610020007); 北京市属高等学校人才强教计划资助项目
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。

关 键 词:李痘病毒  荧光定量RT-PCR  TaqMan探针

Development of a Real-Time Fluorescent Quantitative RT-PCR Method for the Detection of Plum Pox Virus
GAO Ya-Hong,WANG Jin-Zhong,LI Ming-Fu,LI Gui-Fen.Development of a Real-Time Fluorescent Quantitative RT-PCR Method for the Detection of Plum Pox Virus[J].Letters in Biotechnology,2011,22(1):77-80.
Authors:GAO Ya-Hong  WANG Jin-Zhong  LI Ming-Fu  LI Gui-Fen
Institution:GAO Ya-Hong1,2,WANG Jin-Zhong1,LI Ming-Fu2,LI Gui-Fen2 1.Beijing University of Agriculture,Beijing 102206,2.Chinese Academy of Inspection and Quarantine,Beijing 100029,China
Abstract: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 ...
Keywords:Plum pox virus  real-time RT-PCR  TaqMan probe  
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