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任 芳, 张尊平, 范旭东, 胡国君, 张梦妍, 董雅凤*.葡萄卷叶伴随病毒2实时荧光定量RT-PCR技术的检测应用[J].植物保护,2020,46(3):180-187.
葡萄卷叶伴随病毒2实时荧光定量RT-PCR技术的检测应用
Application of a real-time quantitative RT-PCR for detection of Grapevine leafroll-associated virus 2
投稿时间:2019-03-12  修订日期:2019-07-12
DOI:DOI: 10.16688/j.zwbh.2019119
中文关键词:  葡萄  葡萄卷叶伴随病毒2  实时荧光定量RT-PCR  常规RT-PCR  检测
英文关键词:grapevine  Grapevine leafroll-associated virus 2  qRT-PCR  conventional RT-PCR  detection
基金项目:国家重点研发计划(2019YFD1001800);国家现代农业产业技术体系建设专项(CARS-29-bc-1)
作者单位E-mail
任 芳, 张尊平, 范旭东, 胡国君, 张梦妍, 董雅凤* 中国农业科学院果树研究所, 国家落叶果树脱毒中心, 兴城 125100 E-mail:yfdong@163.com 
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中文摘要:
      通过引物筛选和体系优化建立了葡萄卷叶伴随病毒2 Grapevine leafroll-associated virus 2(GLRaV-2)的SYBR Green Ⅰ染料法实时荧光定量RT-PCR检测技术。该技术标准曲线扩增效率为102.2%,决定系数为0.999,最低检出限可达10-3稀释梯度,是常规RT-PCR的100倍。对不同季节和不同部位葡萄样品的检出率普遍高于常规RT-PCR。春夏秋季样品检出率分别为67%、89%和86%,比常规RT-PCR检出率分别高42%、28%和17%。冬季休眠枝条检出率最高(100%),与常规RT-PCR相同。夏季老叶柄和卷须、秋季和冬季枝条等样品检测效果最好,检出率均为100%。对来自我国17个省38个品种的116份田间葡萄样品检测结果表明,qRT-PCR共检测到10个样品为阳性,检出率略高于常规RT-PCR。
英文摘要:
      A SYBR GreenⅠ based real-time quantitative RT-PCR (qRT-PCR) method for Grapevine leafroll-associated virus 2 (GLRaV-2) was established. An excellent linear correlation (0.999) and amplification efficiency (102.2%) were obtained from the standard curve. The detection limit of the method was 10-3 dilution fold, which was 100 times higher than that of conventional RT-PCR. The method was subsequently used to detect grapevine samples in different seasons and different positions of plant. The detection efficiency of qRT-PCR for grapevine samples in most seasons and positions were generally higher than that of conventional RT-PCR. The detection rates of qRT-PCR for samples in spring, summer and autumn were 67%, 89% and 86%, which were 42%, 28% and 17% higher than those of conventional RT-PCR, respectively. For dormant branches in winter, the detection rates were the same for the two methods (100%). In general, the old petioles and tendrils in summer, branches in autumn and winter were the best materials for GLRaV-2 detection by qRT-PCR, with the detection rates of 100%. For field samples (belonging to 38 cultivars) from 17 provinces in China, 10 of 116 samples were detected to be positive by qRT-PCR, and the detection efficiency was higher than that of conventional RT-PCR.
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