水稻干尖线虫的环介导恒温扩增技术(LAMP)快速检测方法

摘要

[目的]本研究旨在为水稻干尖线虫(Aphelenchoides besseyi Christie,1942)的快速检测鉴定和早期诊断提供一个新的检测方法.[方法]利用水稻干尖线虫18S核糖体RNA基因,设计了水稻干尖线虫LAMP的特异引物,该引物包括1对外引物、1对内引物和1对环引物,以LAMP特异引物对待测样品DNA进行恒温扩增,扩增产物通过电泳法和荧光染料法进行检测,电泳检测出现阶梯状条带或加入荧光染料显现绿色荧光,则证明待检样品中含有水稻干尖线虫.[结果]本方法可以检测鉴定水稻干尖线虫不同虫态(雌虫、雄虫、幼虫或卵)的个体,可以从多种线虫混合的样品和植物组织样品中直接检测出水稻干尖线虫,检测灵敏度达到1/1000条虫DNA.[结论]本检测方法具有准确、灵敏、稳定和结果直观的优点,且操作简便、实用性强.%[Objective]This study was performed to provide a new method for the rapid detection and early diagnosis of Aphelenchoides besseyi. [Method]The loop-mediated isothermal amplification (LAMP) specific primers of A. besseyi were designed based on the nematode 18S ribosomal RNA gene, including two external primer, two inner primer and two loop primer. The DNA sample was amplified by LAMP primers using isothermal amplification. The amplified products were detected by electrophoresis and fluorescent dye. It proved that there was A. besseyi in the test sample if there was clear DNA ladder in the electrophoresis results or it showed green fluorescence in the fluorescent dye results. [Result]The results showed that LAMP primers could detect and identify single egg, juvenile, female and male of A. besseyi, and could also directly detect A. besseyi from multiple nematode species samples and plant tissue samples, the sensitivity of LAMP assay was 1/1000 of single nematode DNA. [Conclusion]This diagnosis method has the advantages of accuracy, sensitivity, stability and intuitive results, and is also simple and more practical.