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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。
In order to improve the detection efficiency, nucleic acid was amplified by loop-mediated isothermal amplification (LAMP), and the product was detected by lateral flow dipstick (LFD) A Rapid LAMP-LFD Detection Technique for Meloidogyne camelliae. Three pairs of specific primers were designed to detect the biotinylated real-time fluorescence LAMP reaction using ribosomal DNA-internal transcribed spacer (rDNA-ITS). The optimized LAMP amplification The conditions for the reaction at 63 ℃ for 15 min. Comparison of LFD, real-time fluorescence and agarose gel electrophoresis showed that LAMP product could hybridize with ITS-HP labeled with fluorescein isothiocyanate (FITC) probe for 5 min The color developed on the LFD was only 25 min from the beginning of the LAMP reaction until the LFD result was determined, which was about 2 h shorter than the conventional PCR technique. LAMP-LFD could specifically detect C. elegans, and its detection sensitivity to genomic DNA was 4 pg / μL, which was 100 times lower than that of the conventional PCR method. The detection sensitivity of one second-instar larvae (J2) was 1/1 000 nematodes. The rapid, sensitive and specific LAMP-LFD detection technology established in this study can be applied to the test of root-knot nematodes.