中外葡萄与葡萄酒Issue(5)：26-34,9.DOI:10.13414/j.cnki.zwpp.2026.05.004
基于TaqMan探针的葡萄卷叶伴随病毒-3实时荧光RT-PCR快速检测
TaqMan-Based Real-Time Fluorescence PCR Method for the Rapid Detection of Grapevine Leafroll-Associated Virus-3
摘要
Abstract
To achieve rapid and accurate detection of Grapevine leafroll-associated virus 3(GLRaV-3),in this study,two specific primers(GLRaV-3 F/R)and a TaqMan probe(Probe-GLRaV-3)were designed and synthesized based on the conserved sequence of the coat protein gene of GLRaV-3.The viral positive plasmid standard was prepared,and the TaqMan real-time fluorescent quantitative RT-PCR detection system for GLRaV-3 was established and optimized.The specificity,sensitivity,repeatability and applicability to field samples of this method were systematically evaluated.The results showed that this method could specifically recognize and detect GLRaV-3.The minimum detection limit for the viral positive plasmid standard reached 1.5 copies·μL-1.The established quantitative standard curve had an amplification efficiency of 104.6%and a good linear relationship(R2=0.996),and the detection sensitivity was 100 times that of the conventional RT-PCR method.The method had excellent repeatability,with intra-assay and inter-assay coefficients of variation less than 1.2%and 0.5%,respectively.The optimal primer concentration was 0.3 μmol·L-1,and the optimal annealing temperature was 56℃.The detection results of field grape leaf samples indicated that this method could effectively detect grape leaves infected with GLRaV-3,and its detection sensitivity was higher than that of the conventional RT-PCR method.The detection method established in this study is fast and efficient,with excellent target recognition specificity,outstanding detection sensitivity,and good intra-assay and inter-assay repeatability and stability.It can be used for laboratory detection and field preliminary screening of GLRaV-3,and provides reliable technical support for field identification,early diagnosis and epidemic monitoring of grapevine leafroll disease.关键词
葡萄卷叶伴随病毒-3/实时荧光RT-PCR/TaqMan探针Key words
grapevine leafroll-associated virus-3/real-time fluorescent RT-PCR/TaqMan probe分类
农业科技引用本文复制引用
姚博伟,于泽洋..基于TaqMan探针的葡萄卷叶伴随病毒-3实时荧光RT-PCR快速检测[J].中外葡萄与葡萄酒,2026,(5):26-34,9.基金项目
国家市场监督管理总局科技计划项目(2024MK134) （2024MK134）
宁夏科技惠民项目(2024CMG03049)2023年度宁夏回族自治区青年科技托举人才培养项目(宁科协发组字[2024]6号) （2024CMG03049）
