环介导等温扩增法(LAMP)检测最短尾短体线虫

Detection of Pratylenchus brachyurus by loop-mediated isothermal amplification

为了快速、简便、准确地检测重要植物病原线虫—最短尾短体线虫(Pratylenchus brachyurus),依据GenBank中短体线虫的线粒体DNA细胞色素氧化酶I(mtDNA COI)序列,设计了扩增最短尾短体线虫的环介导等温扩增(loop-mediated isothermal amplification,LAMP)引物,并对LAMP反应条件进行优化,成功建立了一种可特异、快速检测最短尾短体线虫的LAMP体系。该体系在64℃下反应60min,扩增效率最高。用琼脂糖凝胶电泳、酶切分析和SYBR Green Ⅰ染色均能特异检测到最短尾短体线虫的扩增产物。所建立的LAMP体系能从供试的9种短体线虫和另外10种植物线虫中特异检测出最短尾短体线虫,其灵敏度可检测到1/200条线虫DNA,是常规PCR的10倍。表明本研究建立的最短尾短体线虫的LAMP检测体系,可用于最短尾短体线虫的快速检测。

A loop-mediated isothermal amplification(LAMP)assay was developed for rapid and accurate detection of Pratylenchus brachyurus,one of the important plant pathogenic nematodes.Based on the mtDNA COI sequence,LAMP primers for specific detection of P.brachyurus were designed.The optimal conditions of the LAMP assay was the reaction time was 60 min at 64℃.The LAMP products of P.brachyurus were confirmed using conventional polymerase chain reaction(PCR),enzyme digestion analysis with Bam HⅠand visual inspection by adding SYBR Green I to the products.P.brachyurus from nine Pratylenchus species and other ten plant-parasitic nematode species were successfully detected by the LAMP assay.The LAMP assay was also sensitive,which can detect DNA from 0.005 individual P.brachyurus,10 times more sensitive than conventional PCR.The LAMP system constructed in this study can be used to detect P.brachyurus rapidly.