细小种子类毒性药材天仙子的DNA条形码鉴定

Identification of Toxic Hyoscyami Semen and Its Adulterants Using DNA Barcoding

目的:毒性中药的误用滥用对人类健康造成了严重危害。本研究应用DNA条形码技术对细小种子类毒性药材天仙子及其混淆品进行鉴定,为中药材的安全使用提供保障。方法:使用试剂盒提取61份样本的总DNA,通过聚合酶链式反应(PCR)扩增其ITS2序列并双向测序,应用Codon Code Aligner v 4.25对测序峰图进行校对拼接,去除低质量序列及引物区获得ITS2序列。用MEGA 6.0软件计算物种种内和种间Kimura2-Parameter(K2P)遗传距离,分析变异位点并构建邻接(NJ)系统聚类树。结果:天仙子的基原植物莨菪种内最大K2P遗传距离为0.005,远小于其与混淆品之间的种间最小K2P遗传距离(0.360);NJ树结果显示莨菪独自聚为一支,表现出良好的单系性,与混淆品明显区分开。结论:ITS2序列能准确鉴别毒性药材天仙子及其混淆品,为保障临床用药的安全提供了有效的技术手段。

The misuse of toxic drugsisseriousone of the threats of public health. In this study, toxic Hyoscyami Semen and its adulterants were identified by DNA barcoding. The genomic DNA was extracted from 61 samples including Hyoscyami Semen and its adulterants by reagent kit method. 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 Hyoscyamusniger were 0.005 which were smaller than inter -specific ones （0.360） of H. niger and their adulterants. The NJ tree showed that H. niger was clustered into one monophyletic branch, and clearly separated with other species. Therefore, ITS2 sequence was able to identify Hyoscyami Semen and its adulterants to ensure the safty of medicines.