植物罹病组织中南方、爪哇、花生根结线虫的LAMP快速检测

Loop-mediated isothermal amplification assay for rapid diagnosis of Meloidogyne incognita, Meloidogyne javanica and Meloidogyne arenaria from infected plants

【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3种常见的根结线虫,为根结线虫的监测和防治提供技术支持。【方法】分别采用3种根结线虫的种类特异性引物对所选择的根结线虫的DNA片段进行PCR扩增,扩增产物纯化、回收并测序。根据3种根结线虫的测序结果,针对种类特异区段,采用PrimerExplorerV4软件,分别设计3种根结线虫的LAMP引物。设计的引物组人工合成后,以提取的纯化种群线虫DNA为模板,分别进行引物组的特异性测试,筛选出分别针对3种根结线虫的最佳引物组。【结果】研究设计的3种根结线虫的LAMP特异性引物能够直接从植物根结中检测出南方、花生、爪哇3种常见根结线虫,LAMP快速检测体系为:dNTPS浓度为1 mmol·L^(-1),Mg^(2+)的浓度为5 mmol·L^(-1),不添加甜菜碱,反应时间为45 min。【结论】本实验建立的南方、花生、爪哇根结线虫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南方、花生和爪哇根结线虫,具有极高的实践应用价值。

【Aim】 The objective of this study is to establish a rapid diagnostic method for three species of root-knot nematode attacking plants based on the loop-mediated isothermal amplification (LAMP). This could provide technical support for monitoring and preventing the root-knot nematode infestation.【Method】 The DNA fragments of the selected root-knot nematodes were amplified by PCR using species-specific primers of the three root-knot nematodes. The amplified products were purified, recovered and sequenced. Based on the sequencing results of the three root-knot nematodes, three primers were designed using PrimerExplorerV4 software for species-specific segments. The designed primer sets were artificially synthesized, and the purified primer population DNA was used as a template to test the specificity of the primer sets. The best primer sets for three root-knot nematodes were selected.【Result】 The LAMP-specific primers of the three root-knot nematodes could directly detect the three common root-knot nematodes from the roots of plants, LAMP rapid detection system included 1 mmol·L ^-1 dNTPS, 5 mmol·L^-1 Mg^ 2+, without betaine, and at a reaction time of 45 min. The rapid detection of Meloidogyne incognita , Meloidogyne arenaria , Meloidogyne javanica by LAMP was assembled.【Conclusion】 The method was highly specific, sensitive, and economical, which made it possible for quick and accurate detection of M.incognita , M.arenaria and M.javanica from the infected plant root tissues, with high actual application value.