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    烟草靶斑病LFD-RPA快速检测方法的建立

    Establishment of LFD-RPA Rapid Detection Technique for Tobacco Target Spot

    • 摘要: 烟草靶斑病是由立枯丝核菌(Rhizoctonia solani Kühn)侵染引起的烟草叶部病害,近年来在我国部分省份发生严重,危害烟草生产。为开发烟草靶斑病的早期快速诊断方法,本研究根据该病原菌的内转录间隔区(ITS),设计了重组酶聚合酶扩增技术相关引物RsRPA-F3/R3,建立了一种靶斑病菌的重组酶聚合酶扩增结合侧流层析试纸条(LFD-RPA)检测方法。该方法能够在37 ℃,15 min内特异性地检测到R. solani(1.40×102 copies/μL)质粒DNA,与常规PCR灵敏度一致。在田间烟草靶斑病实时检测中,准确率达95.83%。本方法具有快速、简单、可视化等特点,为烟草靶斑病菌的田间快速诊断提供了技术支撑。

       

      Abstract: Target spot caused by Rhizoctonia solani Kühn is a tobacco leaf disease. In recent years, it has occurred seriously in some provinces of China, which endangers tobacco production. In this study, the recombinant enzyme polymerase technology related primers RsRPA-F3/R3 were designed according to the internal transcribed spacer (ITS) of the pathogen, and a method of recombinase polymerase amplification combined with a lateral flow dipstick (LFD-RPA) was developed to establish early and rapid diagnosis technique for tobacco target spot disease. The LFD-RPA detection technology could detect plasmid DNA of R. solani (1.40×102copies/μL) within 15 min at 37 ℃. The sensitivity was consistent with that of conventional PCR. The detection technology can be applied to the real-time detection of tobacco target spot disease in the field with a 95.83% accuracy rate. The rapid detection technology established in this study has the characteristics of fast, simple and visual, and provides a new method for the rapid field diagnosis of tobacco target spot.

       

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