4种葡萄卷叶伴随病毒多重RT-PCR检测  

Detection of four grapevine leafroll-associated viruses by multiplex RT-PCR

作　　者：裴光前[1] 董雅凤[1] 张尊平[1] 范旭东[1] 李丽丽[2] 

机构地区：[1]中国农业科学院果树研究所,辽宁兴城125100 [2]沈阳农业大学园艺学院,沈阳110161

出　　处：《植物病理学报》2010年第1期21-26,共6页Acta Phytopathologica Sinica

基　　金：现代农业产业技术体系专项资金资助(nycytx-30);农业部公益性行业科研专项(nyhyzx07-027)

摘　　要：葡萄受卷叶伴随病毒侵染后,树势减弱,抗逆性变差,果穗着色不良,成熟期推迟,含糖量降低。目前已报道11种葡萄卷叶伴随病毒(Grapevine leafroll-associated virus,GLRaV)。为提高检测效率,降低检测费用,本文在研究单个卷叶伴随病毒RT-PCR检测技术基础上,对4种葡萄卷叶伴随病毒的多重RT-PCR模板浓度、引物浓度和退火温度进行优化,建立了同时检测葡萄卷叶伴随病毒-1(GLRaV-1)、葡萄卷叶伴随病毒-3(GLRaV-3)、葡萄卷叶伴随病毒-4(GLRaV-4)和葡萄卷叶伴随病毒-5(GLRaV-5)的多重RT-PCR技术体系。模板浓度、引物浓度、TaqDNA聚合酶浓度、退火温度和循环次数对多重RT-PCR检测结果均有较大影响,而在一定范围内改变延伸时间和dNTP浓度对检测结果影响较小。对4种葡萄卷叶伴随病毒的PCR产物进行克隆和测序,扩增基因片段与GenBank中登录的基因序列同源性为95%～99%。所建立的多重RT-PCR技术检测田间样品效果良好。The grapevines infected with leafroll viruses were inferior in vigour and stress resistance, poor fruit coloring, delayed maturity and lower sugar content. Eleven grapevine leafroll-associated viruses （GLRaVs） had been isolated from the infected grapevines so far. To improve detecting efficiency and decrease the cost, a multiplex RT-PCR based on single RT-PCR detection of GLRaVs was applied to detecting four grapevine leafroll-associated viruses （GLRaV-1,3,4,5）. In this paper, the mainly factors affecting RT-PCR reaction was studied. The results showed that concentration of template, primer or Taq DNA polymerase, annealing temperature and reaction cycles all had considerable influence on the system, and the influence of extension time or dNTP concentration wasn’t significant. The sequence identity of GLRaV-1 was 96% compared with AF195822, GLRaV-3 was 99% with AJ748524, GLRaV-4 was 99% with EU746619, and GLRaV-5 was 95% with EU815935 in GenBank. The multiplex RT-PCR was proved simple, rapid and sensitive for detection of these 4 GLRaVs.

关 键 词：葡萄卷叶伴随病毒 多重RT-PCR 检测技术 

分 类 号：S432.41[农业科学—植物病理学]