玉米根腐病菌麦根腐平脐蠕孢的LAMP检测方法  

Loop-Mediated Isothermal Amplification Assay for Detection of Bipolaris sorokiniana Causing Maize Root Rot

作　　者：刘树森 郭宁[1] 孙华[1] 马红霞[1] 张海剑[1] 石洁[1] LIU Shusen;GUO Ning;SUN Hua;MA Hongxia;ZHANG Haijian;SHI Jie(Institute of Plant Protection,Hebei Academy of Agriculture and Forestry Sciences,Integrated Pest Management Centre of Hebei Province,Key Laboratory of IPM on Crops in Northern Region of North China,Ministry of Agriculture and Rural Affairs of the People′s Republic of China,Baoding 071000,China)

机构地区：[1]河北省农林科学院植物保护研究所,河北省农业有害生物综合防治工程技术研究中心,农业农村部华北北部作物有害生物综合治理重点实验室,河北保定071000

出　　处：《华北农学报》2023年第1期162-167,共6页Acta Agriculturae Boreali-Sinica

基　　金：国家玉米产业技术体系(CARS-02);河北省重点研发计划(20326501D)。

摘　　要：麦根腐平脐蠕孢是玉米根腐病的重要病原菌之一,旨为建立一种基于环介导等温扩增技术(LAMP)的麦根腐平脐蠕孢快速检测方法。首先,根据麦根腐平脐蠕孢参与黑色素生物合成途径的Brn1基因部分序列设计了一套LAMP检测引物;然后,针对该引物组筛选了其最佳反应温度,检测了LAMP反应的特异性和灵敏度,并以人工接种麦根腐平脐蠕孢的玉米根腐病病株为样本评价了LAMP检测方法的效果。结果表明,本研究设计的引物组在61~68℃条件下均能实现对靶标基因的扩增,其中以66℃为最佳反应温度;在特异性检测中,引物组能从10种分离自玉米根腐病样本的主要病原真菌DNA中特异性地检测出麦根腐平脐蠕孢;在灵敏度检测中,对携带靶基因Brn1的质粒DNA最低检测限是10 copies/μL,在此检测限浓度下,25 min左右即可实现扩增;在对玉米根腐病样本检测中,在1 pg/μL的玉米根组织DNA中即可检测出麦根腐平脐蠕孢。建立的麦根腐平脐蠕孢LAMP检测方法具有较强的特异性和较高的灵敏度。Bipolaris sorokiniana is one of the important pathogens of maize root rot.The purpose of the present work was to establish a rapid detection method for B.sorokiniana based on the loop-mediated isothermal amplification(LAMP).Firstly,a set of primers for LAMP assay was designed according to the partial sequence of Brn1 involved in the melanin biosynthetic pathway.Then,the optimal reaction temperature was screened for this primer set,the specificity and sensitivity of LAMP reaction were detected,and the LAMP detection was evaluated by using the maize root rot samples artificially inoculated with B.sorokiniana.The results showed that the primer set designed could amplify the target gene Brn1 at 61—68℃,with 66℃was the optimal temperature.In the specific detection,the primer set could specifically detect B.sorokiniana from the genomic DNA of 10 main pathogenic fungi isolated from maize root rot plants.In the sensitivity detection,the minimum detection limit for plasmid DNA carrying Brn1 was 10 copies/μL,and amplification could be achieved in about 25 min at the minimum concentration.And,for the detection of maize root rot samples,B.sorokiniana can be detected in 1 pg/μL of maize root tissue DNA.These results indicated that the LAMP detection method for B.sorokiniana established in this study has robust specificity and high sensitivity.

关 键 词：玉米根腐病 麦根腐平脐蠕孢 Brn1基因 环介导等温扩增 检测方法 

分 类 号：S513.03[农业科学—作物学] S435