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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。
Grapevine leaves affected by virus infection, the reduced tenderness, resistance to deterioration, poor ear color, delayed maturation, sugar content decreased. So far, 11 species of Grapevine leafroll-associated virus (GLRaV) have been reported. In order to improve the efficiency of detection and reduce the cost of detection, we studied the single-leaf-based virus by RT-PCR and optimized the template RT-PCR concentration, primer concentration and annealing temperature of four grapevine leaf-associated viruses. (GLRaV-1), GLRaV-3, GLRaV-4 and SCV-5 GLRaV-5) multiple RT-PCR system. The concentration of template, primer concentration, Taq DNA polymerase concentration, annealing temperature and number of cycles had a significant effect on the results of multiple RT-PCR. However, the change of extension time and dNTP concentration within a certain range had little effect on the detection results. The cloned and sequenced PCR products of the four grapevine leaf-associated viruses were 95% -99% identical to those registered in GenBank. The establishment of multiple RT-PCR technology to detect field samples with good results.