三七根腐病菌茄腐镰刀菌的实时荧光LAMP快速检测方法的建立与应用  

Establishment and Application of Real-time Fluorescent LAMP Rapid Detection of Fusarium solani of Panax notoginseng Root Rot

作　　者：车晓莉 梁婷婷 曲媛[1,2] 刘迪秋 CHE Xiao-Li;LIANG Ting-Ting;QU Yuan;LIU Di-Qiu(Faculty of Life Science and Technology,Kunming University of Science and Technology,Kunming 650500,China;Yunnan Key Laboratory of Panax notoginseng,Kunming 650500,China)

机构地区：[1]昆明理工大学生命科学与技术学院,昆明650500 [2]云南省三七资源可持续利用重点实验室,昆明650500

出　　处：《农业生物技术学报》2024年第4期939-948,共10页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金(82060693);云南省重大科技专项计划(202102AA100022)。

摘　　要：根腐病是我国三七(Panax notoginseng)种植区高发的一种严重病害,茄腐镰刀菌(Fusarium solani)是引起三七根腐病的重要病原菌。本研究建立了一套对三七根腐病菌茄腐镰刀菌进行现场快速检测的实时荧光环介导等温扩增(loop-mediated isothermal amplification,LAMP)体系。首先通过PCR反应筛选D-氨基酸氧化酶基因(D-amino acid oxidase gene,DAO)为特异性检测茄腐镰刀菌的基因位点,并对该基因设计LAMP反应的特异性引物组并优化其反应条件,其中65℃为最佳反应温度,MgSO_(4)的最佳浓度为6 mmol/L;dNTPs的最佳浓度为0.6 mmol/L;有环引物存在的情况下,扩增效率更高。分析了LAMP法检测茄腐镰刀菌的灵敏度,模板浓度可低至0.009 pg/μL。最后结合DNA免提法以及钙黄绿素显色法建立了三七根腐病菌茄腐镰刀菌的LAMP现场快速检测技术。本研究为三七种植以及种子种苗交易过程中的根腐致病菌茄腐镰刀菌的快速检测提供技术支撑。Root rot is a serious disease of Panax notoginseng in China,and Fusarium solani is the main fungal pathogen causing root rot of P.notoginseng.In this study,a loop mediated isothermal amplification(LAMP)system was established to rapidly detect F.solani from P.notoginseng.Firstly,the D-amino acid oxidase gene(DAO)was screened by PCR as a specific gene locus for detection of F.solani,and the specific primer pairs of LAMP reaction were designed and the reaction conditions were optimized.The optimum reaction temperature was 65℃;the optimum concentration of MgSO_(4)was 6 mmol/L;the optimum concentration of dNTPs was 0.6mmol/L;the amplification reaction was more efficient in the presence of cyclic primers.Sensitivity of LAMP to F.solani was tested,and the template concentration could be as low as 0.009 pg/μL.At last,the on-site rapid detection technology of F.solani was established based on the combination of DNA extraction-free and calcein coloration.This study provides technical support for the rapid diagnosis of F.solani of root rot in P.notoginseng cultivation and seedling trade.

关 键 词：三七 茄腐镰刀菌 实时荧光LAMP 快速分子检测 

分 类 号：S412[农业科学—植物保护]