《中国药典》中蕲蛇饮片分子鉴定方法的研究和探讨

Study and Discussion on Molecular Identification of Agkistrodon Acutus Medicinal Slices in Chinese Pharmacopoeia

目的:运用多种分子鉴定方法,从技术要求和标准编写方面对蕲蛇饮片现行质量标准提出建议。方法:本研究选取蕲蛇所在蝰科Viperidae蝮亚科Crotalinae的7个近缘物种和9种常见的蕲蛇伪品物种作为研究对象,采用DNA条形码技术扩增16S rRNA序列,运用电泳检视法,根据扩增条带的有无评价模板DNA的质量;16S rRNA序列扩增产物通过双向测序,运用BioEdit软件拼接,双端对齐后剪切比对,利用MEGA 5.1软件构建邻接(NJ)系统发育树;采用蕲蛇饮片现行标准方法进行同步鉴别。结果:16S rRNA序列的电泳检视结果可有效评价蕲蛇样品的模板DNA质量,避免由于模板DNA质量不佳导致的假阴性结果的可能性;基于16S rRNA条形码序列建立的NJ树可以鉴别蕲蛇饮片及其混淆品;蕲蛇饮片现行标准方法专属性良好。结论:DNA条形码方法、现行标准方法以及NJ树三者相互结合,从不同角度对蕲蛇饮片进行真伪鉴别,有效弥补蕲蛇饮片现行标准方法的不足,为其他中药品种的分子鉴定技术的质量标准制定提供了参考。

The study aimed to establish a quality criteria specification for DNA molecular identification of Agkistrodon Acutus medicinal slices through combined use of different molecular identification technologies.Seven relative species in Deinagkistrodon and nine adulterants of Agkistrodon Acutus medicinal slices were selected as test materials.Gel electrophoresis inspection based on DNA barcoding was used to evaluate the quality of template DNA.The 16 S rRNA regions were amplified and sequenced bi-directionally.Sequences that obtained were assembled and aligned by BioEdit.Molecular evolutionary analyses were conducted using MEGA 5.1 to build Neighbor-joining（NJ） phylogenetic tree.Meanwhile,the official identification method was also used in this study.As a result,the quality of template DNA can be evaluated by the results of gel electrophoresis inspection based on DNA barcoding.However,the official identification method was highly specific,which still cannot avoid the misjudgment of false negative.The NJ trees showed that Agkistrodon Acutus can be easily distinguished from its adulterants.Through the combined use of DNA barcoding,official identification method and NJ phylogenetic tree,Agkistrodon Acutus medicinal slices can be accurately identified.Our work also provides an example for accurate molecular identification of quality control.