太子参芜菁花叶病毒和蚕豆萎蔫病毒的双重RT-PCR检测  

Detection of Turnip mosaic virus and Broad bean wilt virus in Pseudostellaria heterophylla by Duplex RT-PCR

作　　者：匡云波[1,2] 陈满足[1] 陆伊荣 陈晶[1] 叶祖云[1,2] 

机构地区：[1]宁德师范学院生物系,福建宁德352100 [2]福建省特色药用植物工程技术研究中心,福建宁德352100

出　　处：《园艺学报》2017年第4期784-791,共8页Acta Horticulturae Sinica

基　　金：福建省教育厅科技项目(JA15563);宁德师范学院引进人才项目(2014Y004);福建省区域发展项目(2015N3014)

摘　　要：针对不同产地栽培的太子参(Pseudostellaria heterophylla)中主要存在芜菁花叶病毒(Turnip mosaic virus,Tu MV)和蚕豆萎蔫病毒(Broad bean wilt virus,BBWV)侵染且为害严重的情况,建立能同时检测这2种病毒的双重RT-PCR快速检测方法。根据Gen Bank数据库中的Tu MV、BBWV外壳蛋白(coat protein,CP)基因核苷酸序列的保守区域分别设计简并引物,在单一RT-PCR检测体系的基础上,建立和优化双重RT-PCR检测体系。双重RT-PCR的优化结果显示,最佳扩增循环数为35,最佳引物浓度为0.4μmol·L^(-1),最佳退火温度为51℃。其灵敏度测定结果显示,2种病毒在样品c DNA稀释至原液的10-4倍后仍能扩增出特异条带。应用该方法对7份栽培太子参样品和4份脱毒苗样品进行了检测,结果表明,建立的双重RT-PCR检测方法可稳定、准确、灵敏地同时检测Tu MV和BBWV。A high incidence of both Turnip mosaic virus（TuMV）and Broad bean wilt virus（BBWV）in cultivated Pseudostellaria heterophylla in different producing areas causes serious damage. A duplex RT-PCR method was developed for simultaneous and rapid detection of those 2 viruses. Degenerate primers were designed according to the conserved sequence regions of coat protein（CP）genes from Gen Bank. Duplex RT-PCR detection system was established and optimized based on single RT-PCR. The optimal parameters for the duplex RT-PCR were 35 PCR cycles,0.4 μmol · L-1 for the concentration of each of the primers,and an annealing temperature at 51 ℃. Sensitivity analysis revealed that viruses could be detected from 104-fold dilution of the first strand c DNA. Duplex RT-PCR established in this study was used to detect 2 viruses in 7 samples of cultivated P. heterophylla and 4 samples of virus-free test-tube plantlets. The results proved that it could be used for rapid detection of TuMV and BBWV simultaneously in P. heterophylla with high stability,accuracy and sensitivity.

关 键 词：太子参 芜菁花叶病毒 蚕豆萎蔫病毒 双重RT-PCR 

分 类 号：S432.41[农业科学—植物病理学] S567[农业科学—农业昆虫与害虫防治]