Rapid and Accurate Detection of the Pathogen Causing Tobacco Target Spot Based on Loop-Mediated Isothermal Amplification  

基于环介导等温扩增技术快速准确检测烟草靶斑病菌

作　　者：LI Ping YIN Xiu-juan WANG Ying ZHOU Xiang-ping LIU Tian-bo ZHONG Jie XIAO Yan-song 黎萍;尹秀娟;王迎;周向平;刘天波;钟杰;肖艳松(永州市农业科学研究所,湖南永州425006;湖南农业大学植物保护学院,湖南长沙410128;湖南省烟草公司永州市公司,湖南永州425000;湖南省烟草科学研究所,湖南长沙410004;湖南省烟草公司郴州市公司,湖南郴州423000)

机构地区：[1]Yongzhou Academy of Agricultural Sciences,Yongzhou 425006,PRC [2]College of Plant Protection,Hunan Agricultural University,Changsha 410128,PRC [3]Yongzhou Branch of Hunan Tobacco Company,Yongzhou 425000,PRC [4]Hunan Tobacco Science Research Institute,Changsha 410004,PRC [5]Chenzhou Branch of Hunan Tobacco Company,Chenzhou 423000,PRC

出　　处：《Agricultural Science & Technology》2024年第4期27-32,共6页农业科技与技术(英文版)

基　　金：湖南省郴州市烟草公司烟草农业科技创新项目(CZYC2022JS03)。

摘　　要：This study established a loop-mediated isothermal amplification(LAMP)system for specially detecting the pathogen causing tobacco target spot in a rapid manner.With genes encoding the Internal Transcribed Spacer(ITS)as targets,four LAMP primers with high specificity and sensitivity were designed and screened.The specificity and sensitivity of the established method were evaluated,and then the method was used to detect the samples of tobacco plants artificially inoculated with pathogens.The established method enabled the observation of detection results by the color change of dye after 60 min of isothermal amplification at 65°C.In the specificity test,only the reaction tubes of the pathogen causing tobacco target spot appeared green,and the electrophoresis lanes were dispersive,indicating positive results,while all other fungal strains showed negative results.The method had a minimum limit of detection being 0.1 ng/μL for the genomic DNA of the pathogen causing tobacco target spot,allowing direct detection of the pathogen from lesions.The established LAMP method can be directly used for the diagnosis of tobacco plants infected with target spot in the field.该研究建立了烟草靶斑病菌的LAMP检测体系,应用该检测体系可实现烟草靶斑病菌的特异性检测及其病害的快速诊断。基于环介导等温扩增技术(Loop-mediated isothermal amplification,LAMP),以编码内转录核酸间隔区(Internal Transcribed Spacer,ITS)的基因作为靶标,设计并筛选了4条特异性强、灵敏度高的LAMP引物。进一步通过特异性和灵敏度检测,以及人工模拟接种和发病组织检测试验,建立了烟草靶斑病菌的LAMP特异性检测体系。该体系经65℃等温扩增60 min后,根据染料颜色变化即可判定扩增结果。在特异性检测中,仅烟草靶斑病菌的反应管呈现绿色,电泳泳道条带呈弥散状态,为阳性结果,而其他真菌菌株均为阴性结果。该方法对于烟草靶斑病菌基因组DNA的最低检测限为0.1 ng/μL,可直接从烟草靶斑病病斑处检测到病原菌。该LAMP检测体系可用于田间烟草靶斑病感病植株的快速诊断。

关 键 词：Pathogen causing tobacco target spot DETECTION Loop-mediated isothermal amplification(LAMP) 

分 类 号：Q94[生物学—植物学]