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园艺学报 ›› 2020, Vol. 47 ›› Issue (4): 797-804.doi: 10.16420/j.issn.0513-353x.2019-0465

• 新技术与方法 • 上一篇    下一篇

杜鹃花枯萎病菌TaqMan MGB探针实时荧光快速检测方法

段丽君1,2,张慧丽1,3,李雪莲1,3,陈先锋1,3,段维军1,3,*   

  1. 1宁波检验检疫科学技术研究院,浙江宁波 315012;2宁波中盛产品检测有限公司,浙江宁波 315012;3宁波海关,浙江宁波 315012
  • 出版日期:2020-04-25 发布日期:2020-04-25
  • 基金资助:
    国家重点研发计划项目(2016YFF0203201);国家留学基金资助项目;浙江省公益技术研究项目(LGF20C140001);宁波市科研项目(2015C110018,2019A610411);宁波中盛产品检测有限公司项目(2019zs06)

Development of TaqMan MGB Fluorescent Real-time PCR Assay for the Detection of Ovulinia azaleae

DUAN Lijun1,2,ZHANG Huili1,3,LI Xuelian1,3,CHEN Xianfeng1,3,and DUAN Weijun1,3,*   

  1. 1Ningbo Academy of Inspection and Quarantine,Ningbo,Zhejiang 315012,China;2Ningbo Joysun Product Testing Service Company,Ningbo,Zhejiang 315012,China;3Ningbo Customs District P. R. China,Ningbo,Zhejiang 315012,China
  • Online:2020-04-25 Published:2020-04-25

摘要: 根据杜鹃花枯萎病菌(Ovulinia azaleae)及其近似种ITS基因序列差异,设计合成1对特异性引物和1条TaqMan-MGB探针,建立了杜鹃花枯萎病菌的实时荧光PCR检测方法。特异性试验结果表明,该检测方法能特异性检测杜鹃花枯萎病菌;灵敏度试验结果表明,最低检测限量为10 μL反应体系中总DNA含量0.25 pg;实际样品检测结果表明,该方法可用于疑似携带杜鹃花枯萎病菌样品的检测与初筛。此方法快速、灵敏,整个反应过程约1 h,检测过程完全闭管,无需PCR后处理,为杜鹃花枯萎病菌早期诊断检测提供了重要参考。

关键词: 杜鹃花, 枯萎病菌, 实时荧光PCR, TaqMan-MGB探针, 检测

Abstract: A precise Real-time polymerase chain reaction(PCR)assay was developed for detection of Ovulinia azaleae in this study. Based on ITS gene of O. azaleae and related isolates,a pair of specific primers and a TaqMan-MGB probe were designed and synthesized. A novel Real-time fluorescent PCR was established to detect O. azaleae. The method detected 0.25 pg of total DNA in 10 μL reaction mixture. The method was rapid,sensitive and completed within a single tube,without post-PCR handling of the amplification products. The new method provides a valuable tool for early rapid detection and identification of O. azaleae.

Key words: Ericaeae, Ovulinia azaleae, real-time fluorescent PCR, TaqMan-MGB probe, detection

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