采用环介导等温扩增法(LAMP)快速检测苹果根结线虫

Loop-mediated isothermal amplification assay for rapid diagnosis of Meloidogyne mali

为高效、简便、快速地对我国进境植物检疫性有害生物名录中的非中国种—苹果根结线虫Meloidogyne mali进行检疫,通过比较Gen Bank中根结线虫相关序列,以苹果根结线虫28S r DNA非保守区域序列设计环介导等温扩增(loop-mediated isothermal amplification,LAMP)的特异性引物,并优化反应条件,建立一种可快速检测苹果根结线虫的LAMP检测体系。结果显示:d NTPs浓度为0.4 mmol/L、Mg^(2+)浓度为5.0 mmol/L、不添加甜菜碱、反应时间为60 min时,LAMP检测体系扩增效率最高;用琼脂糖凝胶电泳、SYBR Green I染色和LFD试纸均能检测到苹果根结线虫的扩增产物。所建立的LAMP检测体系能够从10种供试植物线虫种群中特异性地检测出苹果根结线虫,灵敏度为1/20 000条线虫DNA,比常规PCR灵敏度高10倍。表明所建立的苹果根结线虫LAMP快速检测体系可用于我国口岸进境植物中苹果根结线虫检疫。

In order to efficiently,conveniently and rapidly detect Meloidogyne mali,a non-Chinese species listed in the Catalogue of Quarantine Pest for Import Plants to the People＇s Republic of China,a loop-mediated isothermal amplification（LAMP） assay was carried out.The specific primers for LAMP were designed according to the 28 S ribosomal DNA（r DNA） non-conservative sequences of M.mali by comparing the related sequences of Meloidogyne spp.deposited in Gen Bank.The LAMP amplification with highest efficiency was obtained by the optimized conditions of 0.4 mmol /L d NTPs,5.0 mmol /L Mg^（2＋）,without adding betaine,and 60 minutes of extension.The LAMP products were successfully detected by the agarose gel electrophoresis,SYBR Green I dying and lateral flow dipstick（LFD）.The technique developed in this study was able to specifically detect M.mali from ten species of plant nematodes.The sensitivity of LAMP assay was 1 /20 000 of single nematode DNA,which was ten times higher than that of the conventional PCR assay.The LAMP system established for M.mali detection was proven to be rapid and efficient,and could be further applied in plant quarantine at Chinese ports.