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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。
The results showed that the dNTPS concentration, the Taq enzyme concentration, the primer concentration, the annealing temperature and the amount of the template influencing the multiplex PCR were adjusted and optimized by using the sample of Red Globe grape infected with four viruses in combination. Grapevine leafroll-associated virus-3 (GLRaV-3), Grapevine rupestris stem pitting associated virus (GRSPaV), Grapevine virus B (GVB) and Multiplex RT-PCR method for Grapevine virus A (GVA). Sensitivity test results show that the multiplex RT-PCR and single RT-PCR detection sensitivity basically the same. The specific fragment sizes obtained by multiplex RT-PCR were 905, 546, 460 and 196 bp, respectively. After cloning, sequencing and sequence alignment, the sequences showed high homology with the reported sequences. The results of testing and validation of seven known virus-bearing grapes showed that the established multiplex RT-PCR technique can be used for the detection of these viruses in a large number of field samples.