基于可视化环介导等温扩增技术快速检测中药材蜈蚣真伪

Rapid Detection of Scolopendra Subspinipes Mutilans Using Visual Loop-mediated Isothermal Amplification Technology

目的建立少棘巨蜈蚣的环介导等温扩增(loop-mediated isothermal amplification,LAMP)的方法,以实现对市场蜈蚣药材真伪的快速检测。方法针对少棘巨蜈蚣COI序列设计专属性LAMP引物;考察恒温条件下最佳反应时间;通过对DNA模板的梯度稀释,确定LAMP反应对蜈蚣药材的最低检测浓度;对少棘巨蜈蚣及其伪品进行LAMP扩增反应,以考察LAMP引物对少棘巨蜈蚣的特异性。结果筛选得到了一套特异性较好可用于检测少棘巨蜈蚣的LAMP引物,LAMP反应最佳反应时间为30 min,皮克级别的DNA就能被显著检出。结论LAMP反应对于少棘巨蜈蚣的检测显示了良好的特异性,使用简便,有用于指导市场流通过程中蜈蚣药材的快速检测的潜力。

Objective To establish an identification system of Scolopendra subspinipes mutilans via loop-mediated isothermal amplification(LAMP) method, so as to realize rapid detection of authenticity of Chinese medicine material centipede in the market. Methods We designed specific LAMP primers following the COI gene of S. subspinipes mutilans. Then we inspected the best reaction time at a constant temperature and the lowest detection concentration of DNA template. Finally, to verify the specificity of LAMP primers, we accomplished amplification reaction with S.subspinipes multidens DNA template. Results Our LAMP primers showed a great specificity for detection of S.subspinipes mutilans during a 30 minutes reaction time, and the target DNA could be significantly detected at 10-9mg consistency. Conclusion Scolopendra subspinipes mutilans LAMP reaction showed great potential for the detection,which are able to be applied in the identification of S.subspinipes mutilans as a rapid detection methods in the market.