植物青枯菌LAMP检测方法的建立  

Development of a LAMP Approach for Detection of Ralstonia solanacearum

作　　者：黄雯[1,2] 徐进[2] 张昊[2] 许景升[2] 丁伟[1] 冯洁[2] 

机构地区：[1]西南大学植物保护学院,重庆400716 [2]中国农业科学院植物保护研究所植物病虫害生物学国家重点实验室,北京100193

出　　处：《中国农业科学》2016年第11期2093-2102,共10页Scientia Agricultura Sinica

基　　金：国家自然科学基金(31272008;31371908);国家公益性行业(农业)科研专项(201303015);国家科技支撑计划(2015BAD08B03)

摘　　要：【目的】由茄科雷尔氏菌(Ralstonia solanacearum,简称青枯菌)引起的青枯病(bacterial wilt of plants)是世界范围内危害最为严重的土传细菌病害之一,严重制约了多种经济作物的生产。建立高效、精准的早期诊断技术,是实现青枯病有效防控的基础。论文旨在建立一种能够特异检测青枯菌的环介导等温扩增方法(loop-mediated isothermal amplification,LAMP),实现青枯菌的田间快速检测。【方法】通过比对分析青枯菌的lpx C基因序列,并利用在线引物设计软件Primer Explorer Version 4.0得到4条LAMP特异性引物,F3(5′-CCTGTACGTGGTCGGCTAT-3′)、B3(5′-ACCGCAACACGGGATCA-3′)、FIP(5′-TACGCCGTTTCATCGGCCAGGTACACGGCGCACAAGT-3′)、BIP(5′-ATCGTCACGTTCGACAAGGTGGAATGCCGGCTGCAACTG-3′)。通过单因素变化试验对LAMP反应体系中的各参数进行优化,设置反应温度为60、61、62、63、64、65℃,设置镁离子浓度为2、4、6、8、10、12 mmol·L-1,设置内外引物浓度比为2﹕1、4﹕1、6﹕1、8﹕1、10﹕1、12﹕1,确定最优反应体系。以分离自不同寄主的24个青枯菌株为参试对象,5个非青枯菌株(Ralstonia mannitolilytica、Ralstonia pickettii、Enterobacter sp.、Acidovorax citrulli、Burkhoderia cepacia)为对照,验证LAMP检测方法的特异性。将青枯菌GMI1000菌株的基因组DNA进行10倍梯度系列稀释,以原液和10~1、10~2、10~3、10~4、10~5、10~6、10~7倍的稀释液为模板同步进行LAMP和普通PCR检测,比较两者的检测灵敏度。将马铃薯青枯病菌株Po41、姜青枯病菌株Z-Aq-1分别与马铃薯块茎和生姜根茎组织悬浮液混合,以LAMP检测方法对混合物进行检测,并以同样方法对表现典型萎蔫症状的人工接种番茄植株和健康植株以及田间马铃薯罹病块茎样品进行检测。反应结果直接通过观察产生的白色焦磷酸镁沉淀情况进行判定,或通过加入1μL SYBR GreenⅠ荧光染料进行观察,阳性样品为绿色,阴性样品为橙色。�【Objective】Bacterial wilt of plants, caused by Ralstonia solanacearum, is one of the most devastating soil-borne diseases in the worldwide and severely restricts production of economically important crops. Simple and sensitive detection assay is the basis for effective prevention and control. The objective of this study is to establish a rapid and specific method for detection of R. solanacearum using an isothermal method known as loop-mediated isothermal amplification（LAMP）, to make it possible for researchers and technical staff achieve simple detection of this pathogen. 【Method】 Four specific LAMP primers were designed to target the lpx C of R. solanacearum using online design software Primer Explorer Version 4.0, the inner primers are FIP（5′-TACGCCGTTTCATCGGCCAGGTACACGGCGCACAAGT-3′） and BIP（5′-ATCGTCACGTTCGACAAGGTGGAATGCCG GCTGCAACTG-3′）, the outer primers are F3（5′-CCTGTACGTGGTCGGCTAT-3′） and B3（5′-ACCGCAACACGGGATCA-3′）. Single-factor experiments were conducted to optimize the parameters of the reaction system, the reaction temperatures were set ranging from 60 to 65℃, the concentrations of Mg^（2＋） were set ranging from 2 to 12 mmol·L^（-1）, the concentration ratios of inner and outer primers were set ranging from 2﹕1 to 12﹕1. The specificity of LAMP was tested by using 24 strains of R. solanacearum isolated from different hosts and 5 different strains of non R. solanacearum（Ralstonia mannitolilytica, Ralstonia pickettii, Enterobacter sp., Acidovorax citrulli, Burkhoderia cepacia）, of which 3 species are closely related to R. solanacearum and the others are common bacteria in nature. The sensitivities of LAMP and PCR for detecting R. solanacearum were compared by using ten-fold serially diluted DNA of GMI1000 as templates（including original DNA, 10~1, 10~2, 10~3, 10~4, 10~5, 10~6, and 10~7 times of diluent）. The LAMP method was used to detect the mixture of potato tissue and strain Po41, mixture of ginger tissue and strain Z-Aq-1, also to de

关 键 词：青枯菌 检测 环介导等温扩增 

分 类 号：S432.42[农业科学—植物病理学]