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园艺学报 ›› 2020, Vol. 47 ›› Issue (1): 187-194.doi: 10.16420/j.issn.0513-353x.2019-0216

• 新技术与新方法 • 上一篇    下一篇

葡萄蚕豆萎蔫病毒实时荧光定量RT-PCR检测方法及应用

张梦妍,张尊平,任 芳,胡国君,范旭东*,董雅凤*   

  1. 中国农业科学院果树研究所,国家落叶果树脱毒中心,辽宁兴城 125100
  • 出版日期:2020-01-25 发布日期:2020-01-25
  • 基金资助:
    国家现代农业产业技术体系建设专项资金项目(CARS-29-bc-1);国家重点研发计划项目(2018YFD0201301);中央级公益性科研院所基本科研业务费专项

Establishment and Application of a Real-time Fluorescent Quantitative RT-PCR for Detection of Grapevine fabavirus

ZHANG Mengyan,ZHANG Zunping,REN Fang,HU Guojun,FAN Xudong*,and DONG Yafeng*   

  1. National Center for Eliminating Viruses from Deciduous Fruit Tree,Research Institute of Pomology,Chinese Academy of Agriculture Sciences,Xingcheng,Liaoning 125100,China
  • Online:2020-01-25 Published:2020-01-25

摘要: 建立了葡萄蚕豆萎蔫病毒(Grapevine fabavirus,GFabV)的实时荧光定量RT-PCR(RT-qPCR)检测技术。该技术标准曲线循环阈值与模板浓度呈良好的线性关系(扩增效率103.8%,相关系数0.998),灵敏度是常规RT-PCR的1 000倍,并具有特异性。采用RT-qPCR和常规RT-PCR方法对葡萄不同发育时期及不同部位的316个样品进行检测,结果表明RT-qPCR方法对GFabV的检出率(96.8%)明显高于RT-PCR(70.8%)。RT-qPCR对休眠枝条中GFabV的检出率达100%,对其他部位样品的检出率均在92%以上,明显高于RT-PCR(42% ~ 97%)。各个发育时期样品GFabV的RT-qPCR检出率(85% ~ 95%)也普遍高于RT-PCR(70% ~ 90%)。该技术对于田间样品的检测适用范围广。

关键词: 葡萄, 葡萄蚕豆萎蔫病毒, 实时荧光定量RT-PCR, RT-PCR

Abstract: A SYBR GreenⅠRT-qPCR method for GFabV detection was established,an excellent linear correlation(0.998)and amplification efficiency(103.8%)were obtained from standard curve. The sensitivity of the method was 1 000 times higher than conventional RT-PCR,and had a strong specificity. The established RT-qPCR and conventional RT-PCR were used to detect GFabV in 316 grapevine samples from different development stages and different positions,and the results showed that the detection rate of RT-qPCR(96.8%)was apparently higher than conventional RT-PCR(70.8%). The detection rate of dormant branches using RT-qPCR was 100%,and for other positions detection were all above 92%,which were apparently higher than conventional RT-PCR(42%–97%). The detection rates using RT-qPCR(85%–95%)were higher than conventional RT-PCR(70%–90%)in these samples from different development stages. Compared with conventional RT-PCR,the newly established RT-qPCR has obvious superiority in detection of various samples.

Key words: grape, Grapevine fabavirus, RT-qPCR, RT-PCR

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