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

作　　者：黎妍妍 邱梦娟 李锡宏 张艺千 徐婷婷 许汝冰 马畔 郑露[2] 李伦安 黄俊斌[2] LI Yanyan;QIU Mengjuan;LI Xihong;ZHANG Yiqian;XU Tingting;XU Rubing;MA Pan;ZHENG Lu;LI Lun’an;HUANG Junbin(Tobacco Research Institute of Hubei Province,Wuhan 430030,China;College of Plant Science and Technology,Huazhong Agricultural University,Hubei Provincial Key Laboratory for Crop Disease Monitoring and Safety Control,Wuhan 430070,China;Enshi Marketing Department in Enshi company of Hubei Tobacco company,Enshi,Hubei 445000,China)

机构地区：[1]湖北省烟草科学研究院,武汉430030 [2]华中农业大学植物科学技术学院,湖北省作物病害监测和安全控制重点实验室,武汉430070 [3]湖北省烟草公司恩施州公司恩施市营销部,湖北恩施445000

出　　处：《中国烟草科学》2023年第5期62-69,共8页Chinese Tobacco Science

基　　金：湖北省烟草公司科技项目(027Y2021-004、027Y2022-020);中国烟草总公司重大科技项目[110202101045(LS-05)]。

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(1.40×10^(2) 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。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(1.40×10^(2) copies/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.

关 键 词：烟草靶斑病 立枯丝核菌 重组酶聚合酶扩增技术 侧流层析试纸条 

分 类 号：S435.72[农业科学—农业昆虫与害虫防治]