中成药中全蝎掺伪DNA分子鉴定

Molecular DNA identification of adulteration in processed Chinese herbal medicine containing whole scorpions

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(COⅠ)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1μL,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。

Objective:To establish an effective method for detecting the components of Lychas mucronatus and investigating 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 subunitⅠ(COⅠ)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 annealing temperature of 52℃,36 cycles,2×PCR Mix Taq enzyme,and 1μL of DNA template.The developed method was applied to identify the presence of Centruroides gracilis components in 30 commercially available Chinese 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 control 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 Lychas mucronatus.Phylogenetic analysis indicated that the obtained sequences belonged to the same lineage as Lychas 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 components mixed with authentic scorpion materials in Chinese patent medicines indicates a need for strengthened supervision.The method established in this study enables the rapid and accurate detection of Lychas mucronatus components in Chinese patent medicines.This is beneficial for improving the quality control of whole scorpion-containing Chinese patent medicines,providing assurance for their clinical applications.