中成药中全蝎掺伪DNA分子鉴定

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中文摘要：目的:建立有效检出中成药中细尖狼蝎成分的方法,检测获得中成药中全蝎掺伪情况.方法:收集《中华人民共和国药典》收载的30种含全蝎成分的中成药,提取DNA,比对线粒体细胞色素C氧化酶亚基Ⅰ(CO Ⅰ)基因序列差异,分别设计全蝎源性和细尖狼蝎源性特异性鉴别引物并对退火温度、循环次数、Taq酶以及DNA浓度进行优化,最终确定细尖狼蝎最适PCR鉴别条件包括退火温度52℃,循环数为36,Taq酶为2×PCR Mix,DNA模板为1 μL,通过建立的中成药中细尖狼蝎成分检出方法对市售30种中成药中全蝎投料情况进行鉴定.结果:经凝胶电泳检测,30个全蝎成分中成药均检出正品东亚钳蝎特有DNA条带,18种中成药(60％)在100～250 bp可见单一细尖狼蝎特异性DNA条带,空白对照无条带.对检出细尖狼蝎特异性条带的PCR产物进行TA克隆后测序,序列均与细尖狼蝎参考序列具有很高的相似性(92％～98％),系统发育结果表明获得的序列均与细尖狼蝎为同一支系而与东亚钳蝎不同,因此在120 bp左右出现单一条带可鉴定为细尖狼蝎.结论:中成药中全蝎投料存在以细尖狼蝎掺伪全蝎投料的现象,需要加强监管.本研究建立的方法可以快速准确检出中成药中细尖狼蝎成分,有利于完善全蝎中成药的质量控制方法,为其临床应用提供保障.

外文标题：Molecular DNA identification of adulteration in processed Chinese herbal medicine containing whole scorpions

外文摘要：Objective:To establish an effective method for detecting the components of Lychas mucronatus and in-vestigating instances of adulteration with whole scorpions in Chinese patent medicines.Methods:Thirty Chinese patent medicines containing whole scorpion components,as documented in the Pharmacopoeia of the People's Republic of China,were collected.DNA was extracted,and differences in the mitochondrial cytochrome C oxidase subunit Ⅰ(CO Ⅰ)gene sequences were compared.Specific primers for the identification of Mesobuthus martensii and Lychas mucronatus DNA were designed.Annealing temperature,cycle number,Taq polymerase,and DNA concentration were optimized to establish the most suitable PCR identification system and conditions.The optimal PCR conditions for distinguishing the sharp-tailed wolf scorpion were finally determined to be an an-nealing temperature of 52 ℃,36 cycles,2 × PCR Mix Taq enzyme,and 1 μL of DNA template.The developed method was applied to identify the presence of Centruroides gracilis components in 30 commercially available Chi-nese patent medicines.Results:Gel electrophoresis revealed the presence of authentic DNA bands specific to the Mesobuthus martensii in all 30 whole scorpion-containing Chinese patent medicines.Eighteen medicines(60％)showed a single specific DNA band between 100-250 bp,indicative of Lychas mucronatus,while the blank con-trol exhibited no bands.Sequencing of the PCR products containing the identified Lychas mucronatus-specific bands,after TA cloning,revealed sequences with high similarity(92％-98％)to the reference sequences of Ly-chas mucronatus.Phylogenetic analysis indicated that the obtained sequences belonged to the same lineage as Ly-chas mucronatus and were distinct from the Mesobuthus martensii.Therefore,the presence of a single band around 120 bp indicates the identification of Lychas mucronatus.Conclusion:The presence of Lychas mucronatus compo-nents mixed with authentic scorpion materials in Chinese patent medicines indicates a need for strengthened super-vision.The method established in this study enables the rapid and accurate detection of Lychas mucronatus compo-nents in Chinese patent medicines.This is beneficial for improving the quality control of whole scorpion-contai-ning Chinese patent medicines,providing assurance for their clinical applications.

外文关键词：

Chinese patent medicineButhus martensiiMesobuthus.martensiiLychas mucronatuspolymerase chain reactionmolecular identificationTA clonephylogenetic tree

作者：

段宝丽、尤春雪、蒋超

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作者单位：

道地药材品质保障与资源持续利用全国重点实验室中国中医科学院中药资源中心,北京 100700

天津农学院动物科学与动物医学学院天津市农业动物繁育与健康养殖重点实验室,天津 300392

关键词：

中成药全蝎东亚钳蝎细尖狼蝎聚合酶链式反应分子鉴定 TA克隆系统发育树

基金：

国家自然科学基金项目中国中医科学院中药资源中心自主选题研究项目

项目编号：

83073972ZZXT202212

出版年：

2024

DOI：

10.16155/j.0254-1793.2023-0482

药物分析杂志

中国药学会

药物分析杂志

CSTPCD北大核心

影响因子：1.039

ISSN：0254-1793

年,卷(期)：2024.44(7)

参考文献量11