植物罹病组织中象耳豆根结线虫的LAMP快速检测方法  

Loop-Mediated Isothermal Amplification Assay for Rapid Diagnosis of Meloidogyne enterolobii Directly from Infected Plants

作　　者：何旭峰[1,2] 彭焕[1] 丁中[2] 贺文婷[1] 黄文坤[1] 彭德良[1] 

机构地区：[1]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 [2]湖南农业大学生物安全科技学院,长沙410128

出　　处：《中国农业科学》2013年第3期534-544,共11页Scientia Agricultura Sinica

基　　金：国家公益性行业(农业)科研专项(201103018)

摘　　要：【目的】建立一种基于环介导等温扩增(Loop-mediated isothermal amplification,LAMP)技术,从植物罹病组织中直接检测象耳豆根结线虫(Meloidogyne enterolobii)的快速检测方法,为象耳豆根结线虫的监测和防治提供技术支持。【方法】根据象耳豆根结线虫与其它根结线虫ITS序列差异设计LAMP特异性引物,通过对LAMP反应条件中的Mg2+、dNTPs、甜菜碱和反应时间进行优化,同时对检测体系的特异性和灵敏度进行验证,建立一种从罹病植物组织中检测象耳豆根结线虫的LAMP快速检测方法。【结果】象耳豆根结线虫LAMP检测体系优化结果表明在Mg2+的浓度为5.0 mmol·L-1、dNTPs的浓度为2.4 mmol·L-1、不添加甜菜碱、反应时间为45 min的条件下,扩增效率最优。本研究建立的LAMP检测方法能够从11个植物线虫种群中特异检测出象耳豆根结线虫,检测灵敏度为1/200000头线虫DNA,比普通PCR灵敏100倍,能够直接从植物根结中检测出象耳豆根结线虫,准确度为100%。【结论】本研究以rDNA-ITS序列为靶基因建立象耳豆根结线虫LAMP快速分子检测方法,具有特异性强、灵敏度高、简单、快速、经济等特征,能够从罹病植物组织中快速准确地检测出象耳豆根结线虫,具有极高的实践应用价值。[Objective] The objective of this study is to establish a rapid diagnostic method for Meloidogyne enterolobii from the infected plants based on the loop-mediated isothermal amplification （LAMP）, and to provide technical supports for monitoring and prevention ofM. enterolobii. [Method] The LAMP specific primers were designed according to the distinction of ITS （internal transcribed spacer） sequences between M. enterolobii and other Meloidogyne spp.. The reaction conditions of LAMP, including the concentrations of Mg2＋, dNTPs, betain and reaction times were optimized, and the specificity and sensitivity of the LAMP were testified. A rapid method for M. enterolobii from infected plants by LAMP was established on this basis. [ Result] The optimum conditions for LAMP reaction could be carried out under the concentrations of 5.0 mmol＇L1 Mg2＋ and 2.4 mmol.L1 dNTPs, without betain and 45 minutes of reaction. The method developed in this paper was able to specifically detect M. enterolobii from 11 species of plant nematodes and to sensitivity detect DNA as low as 1/200000 nematode DNA. The sensitivity of LAMP reaction was 100 times higher than the conventional PCR method, which was capable of detecting M. enterolobii directly from the infected roots with 100% accuracy. [ Conclusion] The rapid detection of M. enterolobii by LAMP assay targeted the ITS region of ribosomal DNA was built. The method is high specificity, sensitivity, and economic value, which makes it possible for quick and accurately detecting M. enterolobii from the infected plant tissue, and has higher actual application value.

关 键 词：象耳豆根结线虫 环介导恒温扩增 特异性 灵敏度 快速检测 

分 类 号：S432.45[农业科学—植物病理学]