葡萄4种病毒多重RT-PCR检测体系的建立

Multiplex RT-PCR for Simultaneous Detection of Four Grapevine Viruses

以复合感染4种病毒的‘红地球’(Red Globe)葡萄样品为试材,对影响多重PCR的dNTPS浓度、Taq酶浓度、引物浓度、退火温度及模板量进行了调整和优化,建立了能同时检测葡萄卷叶伴随病毒3(Grapevine leafroll-associated virus-3,GLRaV-3)、沙地葡萄茎痘相关病毒(Grapevine rupestris stem pitting associated virus,GRSPaV)、葡萄病毒B(Grapevine virusB,GVB)和葡萄病毒A(Grapevine virusA,GVA)的多重RT-PCR方法。灵敏度测验结果显示,多重RT-PCR与单一RT-PCR检测灵敏度基本一致。多重RT-PCR获得的特异性片段大小分别为905、546、460和196bp,经过克隆、测序及序列比对,表明其序列与已报道的病毒序列具有较高的同源性。对7个已知带病毒的葡萄样品进行检测验证的结果表明,所建立的多重RT-PCR技术可用于大量田间样品中这些病毒的检测。

Using the‘Red Globe’infected by four grapevine viruses as a sample,the concentrations of dNTPs,Taq DNA polymerase,the primers,annealing temperature and template quantity were optimized for the simultaneous detection of four viruses Grapevine leafroll-associated virus-3（GLRaV-3）, Grapevine rupestris stem pitting associated virus（GRSPaV）,Grapevine virus B（GVB）,Grapevine virus A（GVA） by multiplex RT-PCR. Comparison of the sensitivity of both multiplex-and single RT-PCR for the detection of those viruses showed no significant differences. The target fragments with expected sizes of 905 bp（GRSPaV）,546 bp（GLRaV-3）,460 bp（GVB）and 196 bp（GVA）were amplified from the sample. The multiplex RT-PCR products of those viruses were cloned and sequenced. Result showed that the obtained sequences had high similarities to reported corresponding sequences of those viruses. The viral detection results of seven grapevine samples in field suggested that the multiplex RT-PCR could be used for the simultaneous detection of those four viruses.