含灯盏花中成药DNA提取及其分子鉴定

目的:优选适用于含灯盏花中成药的DNA提取方法和聚合酶链式反应(PCR)扩增引物,并通过测序和系统发育分析实现对其原料灯盏花的分子鉴定。方法:采用4种十六烷基三甲基溴化铵(CTAB)改良方法对3种含灯盏花的中成药进行DNA提取,测定DNA的纯度和浓度。采用内转录间隔区(ITS)、matK、psbA-trnH、rbcL位点通用引物对提取的中成药DNA进行PCR扩增,并对PCR扩增最佳位点进行测序,通过构建系统发育树进行分子鉴定。结果:4种CTAB改良方法均能获得含灯盏花中成药DNA,其中CTAB改良方法一提取的DNA质量浓度显著高于其他改良方法;PCR扩增中以matK位点2对引物(matKXF/matK5R或matK3F/matK1R)最佳,可通过1次PCR成功获得具有单一条带且浓度较高的PCR产物,序列与灯盏花对照药材同源性为100%,系统发育树显示可与同属其他植物区分。结论:通过CTAB改良方法一可以有效提取含灯盏花中成药样品的DNA,采用matK位点引物matKXF/matK5R或matK3F/matK1R进行1次PCR扩增并对产物进行测序,通过序列比对可完成其原料灯盏花的鉴定。

DNA Extraction and Identification of Chinese Patent Drugs Containing Erigeron breviscapus

Objective To optimize the DNA extraction method and polymerase chain reaction (PCR) primers for the molecular identification of the Chinese patent drugs (CPDs) containing Erigeron breviscapus (Vant.) Hand.-Mazz. by sequencing and phylogenetic analysis.Methods Four improved cetyl trimethyl ammonium bromide (CTAB)-based methods were used to extract DNA from three CPDs containing E. breviscapus. The purity and concentration of the extracted DNA were determined, followed by PCR amplification with the general primers at the internal transcribed spacer (ITS), matK, psbA-trnH, and rbcL. The best PCR products were sequenced and used to build the phylogenetic tree for molecular identification.Results Four improved CTAB-based methods could extract DNA from the CPDs containing E. breviscapus, and the concentration of DNA extracted with the improved CTAB-based method 1 was higher than those with other methods (P<0.01). The PCR with primers matKXF/matK5R or matK3F/matK1R demonstrated the best amplification performance, with a unique band and high-concentration PCR product. The product sequence shared the sequence homology of 100% with the standard medical material of E. breviscapus and was in different clades from other species of the same genus in the phylogenetic tree.Conclusion The improved CTAB-based method 1 can extract DNA from the CPDs containing E. breviscapus, and the PCR with the primers matKXF/matK5R or matK3F/matK1R for sequencing and aligning can identify E. breviscapus in CPDs.