橡胶树褐根病病菌LAMP快速检测方法的建立及应用  

Establishment and Application of A Loop-mediated Isothermal Amplification Assay for Rapid Detection of Phellinus noxius on Hevea brasiliensis

作　　者：董文敏 贺春萍[2] 吴伟怀[2] 梁艳琼[2] 谢立 李锐[2] 黄兴[2] 郑金龙[2] 习金根[2] 陆英[2] 易克贤 DONG Wen-Min;HE Chun-Ping;WU Wei-Huai;LIANG Yan-Qiong;XIE Li;LI Rui;HUANG Xing;ZHENG Jin-Long;XI Jin-Gen;LU Ying;YI Ke-Xian(College of Plant Protection,Nanjing Agricultural University,Nanjing 210095,China;Environment and Plant Protection Institute,Chinese Academy of Tropical Agricultural Sciences/Key Laboratory of Integrated Pest Management on Tropical Grops,Ministry of Agriculture and Rural Affairs,China/Hainan Key Laboratory for Monitoring and Control of Tropical Agricultural Pests,Haikou 571101,China;Institute of Forestry,Hainan University,Haikou 570228,China)

机构地区：[1]南京农业大学植物保护学院,南京210095 [2]中国热带农业科学院环境与植物保护研究所/农业农村部热带作物有害生物综合治理重点实验室/海南省热带农业有害生物监测与控制重点实验室,海口571101 [3]海南大学林学院,海口570228

出　　处：《农业生物技术学报》2020年第6期1114-1122,共9页Journal of Agricultural Biotechnology

基　　金：国家重点研发计划项目(2018YFD0201100);国家天然橡胶产业技术体系建设专项资金资助项目(CARS-33-BC1);海南省科协青年科技英才创新计划项目(QCXM201714)。

摘　　要：有害层孔菌(Phellinus noxius)引起的橡胶树(Hevea brasiliensis)褐根病是严重危害天然橡胶的一种根部病害。环介导等温扩增(loop-mediated isothermal amplification,LAMP)技术由于特异性强、灵敏度高、反应迅速、成本低等优势,已在植保等诸多领域得到广泛应用。为了建立一种能够快速、高效、准确地检测橡胶树褐根病菌的技术,本研究以SYBR GreenⅠ为指示剂,建立了橡胶树褐根病菌有害层孔菌的环介导等温扩增可视化快速检测方法。根据橡胶树褐根病菌特异的核糖体转录间隔区序列设计了4条引物(2条内引物FIP/BIP,2条外引物F3/B3)进行LAMP扩增。对LAMP反应条件和体系进行优化,并进行特异性和灵敏度验证,最后进行田间疑似褐根病样本验证。研究确定了最佳反应体系(25μL中模板100 ng/μL,外引物F3/B3各0.2μmol/L,内引物FIP/BIP各1.2μmol/L,dNTPs 1.4 mmol/L,Mg2+6 mmol/L,甜菜碱0.8mol/L,Bst DNA polymerase 8 U/μL)和反应条件(最佳反应温度63℃,反应时间1 h)。特异性验证结果显示,橡胶树褐根病病菌LAMP产物均呈阳性(绿色),而其他病原菌组织LAMP产物均为阴性(橙色)。灵敏度检测结果显示,该体系的DNA最低检测限为1 pg/μL,比常规PCR灵敏度高1000倍。20株田间疑似褐根样本应用LAMP体系检测,其病原检出率为85%,而常规PCR检出率为70%或75%。本研究建立的LAMP检测体系能有效用于橡胶褐根病疑似样本的快速检测,对及时科学的防控该病害具有重要意义。Rubber tree(Hevea brasiliensis)brown root disease caused by Phellinus noxius is a root disease that seriously harms rubber.Loop-mediated isothermal amplification(LAMP)has been widely used in many fields such as plant protection because of its high specificity,high sensitivity,rapid response,and low cost.Therefore,an acturate,sensitive and rapid detection method is a powerful tool to timely effective control measures.In this study,SYBR GreenⅠas an indicator,a fast visual detection method of LAMP for the detection of P.noxius was established.Four primers(2 inner primers FIP/BIP,2 outer primers F3/B3)were designed for LAMP amplification based on the ribosome transcriptional spacer sequence specific to P.noxius.The reaction conditions were optimized and the specificity and sensitivity of LAMP were assayed.The suspected brown root disease samples in the field were verified.The study determined the optimal reaction system(100 ng/μL for template in 25μL,0.2μmol/L for outer primer F3/B3,1.2μmol/L for inner primer FIP/BIP,1.4 mmol/L for dNTPs,and 6 mmol/L for Mg2+,betaine is 0.8 mol/L,Bst DNA polymerase is 8 U/μL and optimal reaction temperature 63°C,reaction 1 h).Specificity assays showed that a positive color(green)was only observed in the presence of P.noxius by SYBR GreenⅠas an indicator reaction prior to amplification,however none of other pathogens changed color(still orange).The lower limit of detection of the LAMP assay was about 1 pg/μL of genomic DNA,which was 1000 times more sensitive than PCR method.Twenty strains of suspected samples in the field were tested by the LAMP system with the pathogen detection rate was 85%,while thenormal PCR detection rate was only 70%or 75%.The LAMP detection system established in this study can be used for the rapid detection of suspected samples of root rot of rubber tree,which was of great significance for effectively controlling the spread of the disease.

关 键 词：橡胶树褐根病 环介导等温扩增(LAMP) 有害层孔菌 快速分子检测 

分 类 号：S432.1[农业科学—植物病理学]