植物罹病组织中南方、爪哇、花生根结线虫的LAMP快速检测

【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。

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

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^-1dNTPS, 5 mmol·L^-1 Mg²⁺, 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.