| 李升,李玲玉,乔光,龙天文,王嘉福.葡萄卷叶病毒3和茎痘病毒双重PCR检测方法的建立及初步应用[J].中国南方果树,2019,48(1): |
| 葡萄卷叶病毒3和茎痘病毒双重PCR检测方法的建立及初步应用 |
| Establishing and Applying the multiplex PCR to detect grapevine leaf roll virus and grapevine rupestris stem pitting-associated virus |
| 投稿时间：2018-10-16 修订日期：2018-10-30 |
| DOI： |
| 中文关键词: 葡萄卷叶病毒3, 葡萄茎痘病毒, 多重PCR |
| 英文关键词:GLRaV-3, GRSPaV, Multiplex PCR |
| 基金项目:本研究由贵州省农业攻关项目(黔科合农NY字(2014)3057号),贵州2015年度千层次创新型人才项目和贵州省2015年度“三区”科技人才项目共同资助。 |
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| 中文摘要: |
| 针对葡萄中卷叶病毒3 (grapevine leaf roll virus 3, GLRaV-3)和茎痘病毒(grapevine rupestris stem pitting-associated virus, GRSPaV)的发生情况,建立了同时检测GLRaV-3和GRSPaV的双重PCR技术,结果表明,建立的双重PCR反应体系最适退火温度为56.1℃,20 μL PCR反应体系中最适引物浓度为0.1 μmol/L；该方法能够检测出GLRaV-3和GRSPaV的cDNA最低浓度分别为0.99 pg/μL和99.43 ng/μL,且特异性良好。应用该双重PCR方法检测了来自贵州凯里和遵义地区的30份不同品种葡萄样品。所测样品中,当地种植的三种葡萄被GLRaV-3的病毒侵染率为100%。将当地检测出的病毒核苷酸序列进行克隆测序后,其中GLRaV-3与美国分离株(AJ748523)相似度最高,为99.82%；GRSPaV与巴西分离株(KT008370)相似度最高,为99.08%。经田间试验,该双重PCR方法可以应用于葡萄GLRaV-3和GRSPaV的检测。 |
| 英文摘要: |
| To investigate the occurrence of grapevine leaf roll virus (GLRaV-3) and grapevine rupestris stem pitting-associated virus (GRSPaV), the multiplex Polymerase Chain Reaction (PCR) method was developed to simultaneous detect the two virus. The results showed that the optimum annealing temperature of the multiplex PCR reaction system was 56.1°C and the optimum primer concentration was 0.1 μmol/L. The minimum detectable concentration of GLRaV-3 cDNA and GRSPaV cDNA were 0.99 pg/μL and 99.43 ng/μL separately when using the multiplex PCR method. Further, this method had also a high specificity, with no cross reaction to other grapevine leaf roll virus. 30 different grapes from Kaili, Guizhou Province were detected by the multiplexPCR method and the result showed all the Wink grapes were infected by the GLRaV-3. The nucleotide sequences of GLRaV-3 and GRSPaV were isolated in the Kaili and Zhunyi area. After compared with the virus from other areas, the sequence of GLRaV-3 had a highest identity (99.82%) with the American isolated strain (AJ748523), the GRSPaV had the highest similarity (99.08%) with the Brazilian isolated strain (KT008370). In conclusion, the field experiment results indicated this multiplex RT-PCR method could effectively simultaneous detect the GLRaV-3 and GRSPaV. |
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