烟草环斑病毒的定量检测技术研究  

Detection of Tobacco Ringspot Virus by Real-Time RT-PCR Assay

作　　者：刘梅[1,2] 黄新[1] 马占鸿[2] 陈洪俊[1] 李明福[1] 

机构地区：[1]中国检验检疫科学研究院动植物检疫实验所,北京100029 [2]中国农业大学植物检疫系,北京100094

出　　处：《生物技术通讯》2010年第1期73-76,共4页Letters in Biotechnology

基　　金：国家质检总局基金(2006IK209);国家"十一五"科技支撑计划(2006BAD08A16-1)

摘　　要：目的:建立特异、灵敏、快速的TaqMam实时荧光定量PCR方法,用于烟草环斑病毒(TRSV)的定量检测。方法:用纳米磁珠法提取病毒RNA,构建包含烟草环斑病毒全CP序列的质粒标准品。根据CP保守序列设计特异性的引物和TaqMam荧光探针,构建标准曲线,建立TRSV的实时荧光绝对定量PCR方法,并对该方法的特异性、灵敏度和重复性进行评估。结果:建立的方法特异性好,与南芥菜花叶病毒、马铃薯X病毒和马铃薯Y病毒均无交叉反应;至少能检测到767个病毒拷贝,灵敏度比普通PCR高100倍;同一样品试验内及试验间重复性实验的变异系数均小于3%,重复性好;检测结果准确可靠,构建的标准曲线有较好的线性关系(R2=0.997)。结论:建立的TRSV TaqMan实时荧光定量PCR检测方法可满足口岸高通量、快速、准确的检验检疫要求。Objective：To establish a TaqMan-based real-time PCR assay for detection and quantification of Tobacco ringspot virus（TRSV）.Methods：The virus RNA was extracted using viruses RNA isolation system based on magnetic nano-particles.The method was designed to use specific primers and TaqMan probe targeting conserved sequence in the coat protein gene region of TRSV down-loaded from GenBank.The specificity,sensitivity and reproducibility of the method were estimated and a standard curve was prepared.Results：The specificity of the assay was high and there were no cross reactions with Arabis mosaic virus,Potato virus X and Potato virus Y.The sensitivity of the assay was 767 plasmid copies and it＇s 100 fold sensitive than conventional PCR.Both coefficients of variation were less than 3%,indicating a good reliability.A good linear correlation was demonstrated（linear regression R2=0.997）.Conclusion：This TaqMan-based real-time PCR assay is a quick,sensitive and specific tool for molecular diagnosis of Tobacco ringspot virus.It could be applied to exit-entry quarantine detection for its high throughput,rapidness and precision.

关 键 词：烟草环斑病毒 实时荧光定量PCR TAQMAN探针 

分 类 号：Q78[生物学—分子生物学]