快速检测中药材乌梢蛇LAMP方法的建立

乌梢蛇作为一种名贵中药材,市面上伪品较多,干燥熏黑处理后的样品,更是真伪难辨。本研究致力于开发一套基于环介导等温扩增(loop-mediated isothermal amplification,LAMP)为基础的快速筛查乌梢蛇的方法。本研究以乌梢蛇12srRNA基因序列为基础设计并筛选出1套LAMP引物。通过调整反应条件,建立了对乌梢蛇LAMP的检查方法。结果显示,62℃下连续反应15min左右出现典型的"S"型荧光吸收曲线,实现了对乌梢蛇12srRNA基因序列的特异扩增。根据LAMP灵敏度高的特点,本研究简化了DNA的提取方法,缩短了检测的时间。相对于常规的PCR方法,本研究建立的以快速DNA提取为基础的乌梢蛇LAMP快速筛查方法具有简单、快速、灵敏、对设备要求低等特点,适用于对中药材乌梢蛇的快速筛查。

Establishment of Loop-Mediated Isothermal Amplification Method for Rapid Screening of Zaocys dhumnades

Zaocys dhumnades is a kind of valuable Chinese herbal medicine.There are many kinds of fakes on the market and it is very hard to distinguish the real from the fake,especially when the snakes are treated by drying and fumigation.In order to develop a rapid screening method based on loop-mediated isothermal amplification(LAMP),proper primers for LAMP were designed according to the 12 srRNA gene of Zaocys dhumnades,and determined by screening experiments.By adjusting the reaction conditions,the LAMP method for specific amplification of Zaocys dhumnades was established.The LAMP reaction result showed that a typical"S"curve of fluorescence absorption occurred when the LAMP reaction lasted for 15 minutes at 62 ℃.According to the characteristics of high sensitivity of LAMP,this study simplified the extraction method of DNA and shortened the whole detection time.Compared with conventional PCR method,the rapid screening method of LAMP combined with rapid DNA extraction is simple,quick,highly sensitive,and low requirement for the equipment.This study shows that the LAMP method can be applied to the rapid screening of Zaocys dhumnades.