Rapid detection of Meloidogyne spp. by LAMP assay in soil and roots
Rapid detection of Meloidogyne spp. by LAMP assay in soil and roots
复制标题
快速检测根结线虫属。
DOI：
10.1016/j.cropro.2011.03.028
复制
发表时间：
2011-08-01
期刊：
影响因子：
2.8
通讯作者：
Guo, Yang-dong
中科院分区：
文献类型：
--
作者：
Niu, Jun-hai;Guo, Quan-xin;Guo, Yang-dong
环介导等温扩增（LAMP）是一种新型的DNA扩增技术，已被用于检测包括病毒、真菌、细菌和寄生虫在内的多种病原体。然而，尚未尝试诊断定居植物寄生线虫（PPN）物种。本研究建立了一套用于诊断南方根结线虫（Meloidogyne incognita）、南方根结线虫（M. arenaria，M. javanica和M. hapla）和M.无名氏特异性LAMP集（Mi-LAMP）。在两种试验中，在所有阳性样品中均观察到凝胶电泳上的典型梯状模式，但在阴性对照中未观察到。使用Hpa II酶的限制性分析、通过使用SYBR绿色I的目视检查和侧流试纸（LFD）测定法进行检测来进一步确认扩增产物。这两套LAMP系统能够特异性地检测四种常见的根结线虫和M.有几个不同的地理起源和致病型的未知种群。与其它PPN的DNA无交叉反应。RKN-和Mi-LAMP的灵敏度分别为10和100 fg的纯基因组DNA。这两套LAMP引物也能扩增出从根组织和土壤中提取的粗DNA。无名氏RKN和Mi-LAMP系列具有简单、快速和成本效益的优点。这两套LAMP将有助于诊断根结线虫属。当地推广和监管人员。(C)2011爱思唯尔有限公司保留所有权利。
Loop-mediated isothermal amplification (LAMP), a novel DNA amplification technique, has been used to detect a variety of pathogens including viruses, fungi, bacteria and parasites. However, diagnosis of sedentary plant-parasitic nematode (PPN) species has not yet been attempted. In this study, we developed a universal LAMP set (RKN-LAMP) for the diagnosis of four common Meloidogyne species (Meloidogyne incognita, M. arenaria, M. javanica and M. hapla), and M. incognita-specific LAMP set (Mi-LAMP). In both assays, a typical ladder-like pattern on gel electrophoresis was observed in all positive samples but not in the negative controls. Amplification products were further confirmed using restriction analysis of the Hpa II enzyme, detection by visual inspection using SYBR Green I and the lateral flow dipstick (LFD) assay. The two LAMP sets were specifically able to detect four common Meloidogyne species and M. incognita populations having several different geographical origins and pathotypes. No cross reaction with DNA of other PPNs was observed. Sensitivity of the RKN- and Mi-LAMP was 10 and 100 fg of pure genome DNA respectively. Both LAMP sets could also amplified crude DNA isolated from the galled root tissue and from soil containing juveniles of M. incognita. The RKN- and Mi-LAMP sets offer the advantages of simplicity, rapidity and cost effectiveness. Both LAMP sets will be instrumental for the diagnosis of Meloidogyne spp. by local extension and regulatory personnel. (C) 2011 Elsevier Ltd. All rights reserved.