基于ITS2序列鉴定谷物芽类药材及其混伪品

目的：应用ITS2序列对2010版《中国药典》谷物芽类药材稻芽、谷芽、麦芽及其混伪品进行分子鉴定，以保证药材质量和临床疗效。方法：提取稻芽、谷芽和麦芽药材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软件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MEGA6.0软件对所有10个物种74条序列计算种内和种间K2P（Kimura 2-Parameter）遗传距离，分析变异位点并构建邻接（NJ）树。结果：稻芽、谷芽和麦芽的基原植物稻、粟和大麦的种内最大K2P遗传距离均远小于其与混伪品之间的种间最小K2P遗传遗传距离；NJ树结果显示稻、粟和大麦各自聚为一支，均与混伪品明显区分开，各混伪品物种也单独聚为一支。结论：ITS2序列能准确鉴别稻芽、谷芽、麦芽药材及其混伪品，该研究为保障谷物芽类药材临床准确用药提供了新的技术手段。

Identification of Grain Sprouts Herbs and Their Adulterants Using ITS2 Barcode

Some grain sprouts herbs recorded in Chinese Pharmacopoeia 2010, including Oryzae Fructus Germinatus, Setariae Fructus Germinatus and Hordei Fructus Germinatus, together with their adulterants, were identified by ITS2 sequences. The genomic DNA were extracted, their ITS2 sequences were amplified, and purified PCR products were sequenced. Sequences were assembled using the CodonCode Aligner. The genetic distances, variable sites and the neighbor-joining (NJ) phylogenetic tree were computed by MEGA 6.0 in accordance with the Kimura 2-Parameter (K2P) model. The results showed that their intraspecific genetic distances were all much lower than the interspecific ones of themselves and their adulterants. The NJ tree based on ITS2 sequence indicated that Oryza sativa, Setariaitalica, and Hordeum vulgar formed one monophyletic clade respectively, in addition any one of the grain sprouts herbs and its adulterants could be distinguished clearly. Therefore, ITS2 sequence is suitable to be as a barcode to identify the grain sprouts herbs and their adulterants.