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目的采用DNA条形码分子鉴定技术鉴别市售柴胡药材及其混伪品,以确保柴胡药材质量及临床用药安全。方法共收集85份柴胡药材,对其ITS2序列进行PCR扩增并双向测序,所得序列经CodonCode Aligner校对拼接后,利用MEGA 6.0对序列进行分析比对,计算种内、种间遗传距离,利用邻接法(NJ)构建系统聚类树,并应用ITS2数据库网站预测其ITS2二级结构。结果柴胡种内遗传距离明显小于柴胡与其近缘物种种间遗传距离,基于ITS2建立的NJ树和网站预测的ITS2二级结构,均能将柴胡药材及其混伪品区分开。85份样品中,55份符合《中国药典》2015年版规定的柴胡正品来源,正品率为64.7%。结论基于ITS2序列可以准确可靠地鉴别柴胡药材及其混伪品,为确保临床用药安全提供新的技术手段。
Objective To identify the quality of Bupleurum medicinal materials and their counterfeit products by using DNA barcode molecular identification technology to ensure the quality of Bupleurum medicinal materials and the clinical medication safety. Methods A total of 85 Bupleurum medicinal materials were collected and their ITS2 sequences were amplified by PCR and sequenced. The sequences were aligned by CodonCode Aligner and analyzed by MEGA 6.0. The genetic distances between species and species were calculated. Adjacent method (NJ) was used to construct phylogenetic tree and ITS2 secondary structure was predicted by ITS2 database. Results The genetic distance of Bupleurum chinense L. was significantly smaller than that of Bupleurum chinense and its related species. Based on NJ tree established by ITS2 and ITS2 secondary structure forecasted by the website, both Bupleurum chinense and its adulterants can be distinguished. Of the 85 samples, 55 were genuine sources of Bupleurum rooted in the 2015 edition of the Chinese Pharmacopoeia, with a genuine rate of 64.7%. Conclusion Based on the ITS2 sequence, the Bupleurum medicinal materials and their counterfeit products can be accurately and reliably identified, providing new technical means for ensuring the safety of clinical medication.