烟草环斑病毒RT-PCR检测体系的优化  

Optimization of RT-PCR Detection System for Tobacco Ringspot Virus

作　　者：郇晓雪 刘晓宇 房乐[1] 杜传印 李广强 耿超 李向东[1] 田延平[1] Huan Xiaoxue;Liu XiaoYu;Fang Le;Du Chuanyin;Li Guangqiang;Geng Chao;Li Xiangdong;Tian Yanping(College of Plant Protection,Shandong Agricultural University,Taian 271018,China;Shandong Weifang Tobacco Company,Weifang 261061,China;Zaozhuang Agriculural and Rural Derelopment Center,Zaozhuang 277899,China)

机构地区：[1]山东农业大学植物保护学院,山东泰安271018 [2]山东潍坊烟草公司,山东潍坊261061 [3]枣庄市农业农村事业发展中心,山东枣庄277899

出　　处：《山东农业科学》2021年第6期98-102,共5页Shandong Agricultural Sciences

基　　金：国家自然科学基金项目(31871933,31501612);山东省烟草公司重大项目(鲁烟科〔2021〕3号);潍坊市烟草公司项目(潍烟技〔2021〕2号-2021-47)。

摘　　要：烟草环斑病毒(Tobacco ringspot virus,TRSV)是豇豆花叶病毒科(Comoviridae)线虫传多面体病毒属(Nepovirus)的代表种,是我国公布的二类进境检疫有害生物。建立特异性强、灵敏性高的检测体系是有效控制TRSV传播的技术保障。本研究在分析引物的特异性、优化退火温度及dNTPs、引物、Taq DNA聚合酶浓度的基础上,建立了完善的TRSV RT-PCR检测体系。筛选出的TRSV特异性引物对为1-1F/1-1R,在退火温度为53℃、dNTPs终浓度为0.075 mmol/L、引物终浓度为0.30μmol/L、Taq DNA聚合酶终浓度为0.035 U/μL条件下检测效率最高,可从发病植物总RNA量为0.090 ng中检测到目的片段。研究结果对准确灵敏地检测TRSV具有重要意义。Tobacco ringspot virus(TRSV)is the type member of genus Nepovirus in the family Comoviridae,and is a second-class quarantine pest in China.Specific and accurate detection is essential to effectively prevent TRSV spread.In this study,we screened specific primers,optimized the annealing temperature and the concentrations of dNTPs,primers and Taq DNA polymerase,and established an optimized RT-PCR detection system for TRSV.The results showed that the primer pair 1-1 F/1-1 R had the highest amplification efficiency.The detection efficiency was the highest under the conditions of 53℃of annealing temperature,0.075 mmol/L dNTPs,0.30μmol/L primers and 0.035 U/μL Taq DNA polymerase,which could detect TRSV from 0.090 ng total RNA of TRSV-infected tobacco plants.The results were of great significance to accurate and sensitive detection of TRSV.

关 键 词：烟草环斑病毒 检测体系 逆转录聚合酶链式反应(RT-PCR) 优化 

分 类 号：S435.72[农业科学—农业昆虫与害虫防治]