山茶根结线虫的LAMP-LFD快速检测方法  

Rapid and Sensitive Detection of Meloidogyne camelliae by LAMP-LFD

作　　者：蔡怡[1] 周前进[1] 顾建锋 陈先锋[1,2] 陈炯[1] 

机构地区：[1]宁波大学生物与海洋科学系,宁波315211 [2]宁波检验检疫科学技术研究院,宁波315012

出　　处：《农业生物技术学报》2016年第5期770-780,共11页Journal of Agricultural Biotechnology

基　　金：国家科技支撑计划项目(No.2012BAK11B03);浙江省自然科学基金项目(No.LY14C180002);宁波市科技创新团队(No.2015C110018);宁波大学2015年度研究生科研创新基金(No.G15065)

摘　　要：为了提高检测效率,本研究利用环介导等温扩增技术(loop-mediated isothermal amplification,LAMP)扩增核酸,用横向流动试纸条(lateral flow dipstick,LFD)检测产物,建立了一种山茶根结线虫(Meloidogyne camelliae)的LAMP-LFD快速检测新技术。以山茶根结线虫核糖体DNA内转录间隔区(ribosomal DNA-internal transcribed spacer,r DNA-ITS)为检测靶标,设计3对特异性引物进行生物素标记的实时荧光LAMP反应,优化后的LAMP扩增条件为63℃反应15 min。比较LFD、实时荧光曲线以及琼脂糖凝胶电泳等3种产物检测方法,结果表明,LAMP产物与异硫氰酸荧光素(fluorescein isothiocyanate,FITC)标记的探针ITS-HP杂交5 min后即可在LFD上显色,从LAMP反应开始到LFD结果判断仅需25min,比常规PCR技术缩短约2 h。LAMP-LFD技术能特异性地检测山茶根结线虫,对其基因组DNA的检测灵敏度为4 pg/μL,低于常规PCR方法100倍;对其单条2龄幼虫(J2)检测灵敏度为1/1 000条线虫。本研究建立的快速、灵敏、特异性LAMP-LFD检测技术可应用于山茶根结线虫的口岸检验。Aiming at improving the detection efficiency, a novel loop-mediated isothermal amplification- lateral flow dipstick （LAMP-LFD） method for rapid detection of Meloidogyne camelliae was developed, mostly based on the nucleotide amplification by a LAMP and visual detection by a LFD. Three pairs of primers were designed targeting the conserved region of ribosomal DNA-internal transcribed spacer （rDNA- ITS） and used in the biotinylated Real-time fluorescence LAMP. The optimized LAMP was of 63 ~C 15 min. LFD, Real-time fluorescence curve and agarose gel electrophoresis were separately used for LAMP products detection, and the practicability of LFD was comparatively evaluated. The results indicated that LAMP products were hybridized by a fluorescein isothiocyanate （FITC）-labeled probe ITS-HP and visually detected in LFD in 5 min. It just needed 25 min from the beginning of LAMP reaction to the judgment of results, which was approximately 2 h time-savings compared with the conventional PCR method. The results demonstrated that LAMP-LFD could specifically detect M. camelliae with a detection limit of 4 pg/μL M. camelliae genomic DNA, which was lower than the conventional PCR of 100 times, and the detection sensitivity was 1/1 000 for a single juvenile J2 genomic DNA. Therefore, this rapid, sensitive and specific LAMP-LFD was an expected option in the inspection and quarantine for M. camelliae.

关 键 词：山茶根结线虫 环介导等温扩增(LAMP) 横向流动试纸条(LFD) 检测 

分 类 号：S432.45[农业科学—植物病理学]