基于ITS2序列鉴定南北葶苈子药材及其混伪品

摘要

目的：利用ITS2序列对多基原药材葶苈子及其混伪品进行分子鉴定，以保证药材质量及临床疗效。方法：提取46份葶苈子药材及其混伪品的DNA ，通过聚合酶链式反应（PCR）扩增其ITS2序列并双向测序，应用CodonCode Aligner v 4.25对测序峰图进行校对拼接，并去除低质量序列及引物区，得到ITS2序列。用MEGA 6.0软件计算物种种内和种间 Kimura 2-parameter（K2P）遗传距离，分析变异位点并构建邻接（NJ）系统聚类树，综合应用相似性搜索法、最近距离法以及NJ系统发育树等进行鉴定分析。结果：葶苈子的基原植物播娘蒿和独行菜的种内最大K2P遗传距离分别为0.021和0.010，均小于其与混伪品之间的种间最小K2P遗传距离；NJ树结果显示播娘蒿和独行菜各自聚为一支，表现出良好的单系性，均可与混伪品明显区分开。结论：以上几种鉴定方法分析结果表明，ITS2序列能准确鉴别南北葶苈子药材与混伪品，为保障临床安全用药提供了新的技术手段。%Descurainiae, Lepidii Semen and their it adulterants were identified by analysising their ITS2 sequences. The genomic DNA was extracted from 46 samples including Descurainiae and Lepidii Semen and their it adulterants. Their ITS2 sequences were amplified, and purified PCR products were sequenced. Sequence assembly and consensus sequence generation were performed using CodonCode Aligner v 4.25. The genetic distances, variable sites and the neighbor-joning (NJ) phylogenetic tree were computed by MEGA 6.0 in accordance with the Kimura 2-parameter (K2P) model. The results showed that the intra-specific genetic distances of Descurainia sophia and Lepidium apetalum were 0.021 and 0.010, which were smaller than inter-specific ones of D. sophia, L. apetalum and their adulterants. The NJ tree showed that both D. sophia and L. apetalum were clustered into one monophyletic branch, and clearly separated with their sibling species. Therefore ITS2 sequence was able to identify Descurainiae and Lep-idii Semen and its adulterants to ensure the quality of medicines and clinical efficacy.