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园艺学报 ›› 2020, Vol. 47 ›› Issue (2): 390-398.doi: 10.16420/j.issn.0513-353x.2019-0566

• 新技术与新方法 • 上一篇    下一篇

基于重组酶聚合酶扩增(RPA)技术的樱桃病毒A(CVA)的检测方法

陈 玲,段续伟,张开春*,张晓明,王 晶,闫国华,周 宇   

  1. 北京市林业果树科学研究院,农业农村部华北都市农业重点实验室,农业农村部华北地区园艺作物生物学与种质创制重点实验室,北京 100093
  • 出版日期:2020-02-25 发布日期:2020-02-25
  • 基金资助:
    国家自然科学基金青年基金项目(31601726);农产品质量与农田环境基础数据平台建设项目(xKJCX20170302);北京市农林科学院协同创新专项(KJCX201907-2,KJCX201910)

A Method for the Detection of cherry virus A(CVA)Based on Recombinase Polymerase Amplification(RPA)Technique

CHEN Ling,DUAN Xuwei,ZHANG Kaichun*,ZHANG Xiaoming,WANG Jing,YAN Guohua,and ZHOU Yu*   

  1. Key Laboratory of Urban Agricultural(North China),Ministry of Agriculture and Rural Affairs;Key Laboratory of Biology and Genetic Improvement of Horticultural Crops(North China),Ministry of Agriculture and Rural Affairs;Beijing Academy of Forestry and Pomology Sciences,Beijing 100093,China
  • Online:2020-02-25 Published:2020-02-25

摘要: 根据樱桃病毒 A(cherry virus A,CVA)外壳蛋白基因序列的保守区设计特异性引物和探针,建立了樱桃中 CVA 的重组酶聚合酶扩增(recombinase polymerase amplification,RPA)检测方法。该方法可在恒温 38 ℃条件下进行,40 min 即可完成检测。灵敏度试验表明,采用 RPA 方法检测 CVA 的灵敏性是 PCR 方法的 10 倍,且与侵染樱桃的另外 6 种病毒和 1 种类病毒无交叉反应。此外,具有标记的扩增子可通过侧向流试纸条直接观察结果。采用该方法检测田间样品效果良好。

关键词: 樱桃, 樱桃病毒 A, 重组酶聚合酶扩增, 检测

Abstract: In this study,recombinase polymerase amplification(RPA)method was developed using the specific primers and probe designed based on the conserved gene sequences of cherry virus A(CVA)coat protein to detect CVA in cherry. The RPA method could be conducted under thermostotic condition at 38 ℃ and was completed within 40 min. The sensitivity test showed that the RPA was about 10-fold more sensitive than PCR and no cross-reactivity was detected with other 6 cherry-infected viruses and 1 viroid in China. Moreover,the terminally labeled amplicons could be visually inspected with lateral flow strip. This method was verified effectively by testing field-collected samples.

Key words: cherry, cherry virus A(CVA), recombinase polymerase amplification(RPA), detection

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