土鳖虫(地鳖)配方颗粒的特异性PCR鉴别研究  

Identification of Eupolyphaga Steleophaga(Eupolyphaga sinensis)formula granules by specific PCR

作　　者：刘闪闪 谭斯尹 宋叶 冼乐尧 范耀耀 罗宇琴 李国卫 LIU Shan-shan;TAN Si-yin;SONG Ye;XIAN Le-yao;FAN Yao-yao;LUO Yu-qin;LI Guo-wei(Guangdong Yifang Pharmaceutical Co.,Ltd.,Guangdong Key Laboratory of TCM Formula Granule Enterprises,Foshan 528244,China)

机构地区：[1]广东一方制药有限公司广东省中药配方颗粒企业重点实验室,佛山528244

出　　处：《药物分析杂志》2024年第11期1951-1957,共7页Chinese Journal of Pharmaceutical Analysis

摘　　要：目的:建立基于SNP位点的特异性PCR技术对土鳖虫(地鳖)配方颗粒及其混伪品的分子鉴定方法。方法:通过分析土鳖虫与其常见伪品的细胞色素C氧化酶亚基1(COⅠ)序列,寻找土鳖虫SNP变异位点,设计出特异性引物TBC-F(5’-TTCTTGTTGGCAAGCAGTATAAT-3’)和TBC-R(5’-AACTACTGCTCAAACAAATAATGGA-3’)。采用三步法进行聚合酶链式反应(PCR),并通过方法优化,确立最佳反应体系,同时为保证试验结果的准确性,将PCR扩增产物进行一代测序。结果:在退火温度为55~57℃,循环数为37次的扩增条件下,21批土鳖虫药材、20批标准汤剂及其19批配方颗粒经PCR扩增及凝胶电泳检测后,在237 bp处有单一明亮的特异性条带,其伪品无条带,试验结果与一代测序结果一致,为地鳖。结论:建立的特异性PCR鉴别方法可准确对土鳖虫药材、标准汤剂冻干粉、配方颗粒成品进行鉴别,为土鳖虫的配方颗粒质量标准研究提供了参考依据。Objective:To establish a specific polymerase chain reaction(PCR)method based on SNP loci for molecular identification of Eupolyphaga Steleophaga formula granules and its counterfeit.Methods:By analyzing the cytochrome oxidaseⅠ(COⅠ)sequences of Eupolyphaga Steleophaga and its common forgeries,the SNP mutation sites of Eupolyphaga Steleophaga were searched and specifi cprimers were designed,which were TBC-F(5'-TTCTTGTTGGCAAGCAGTATAAT-3')and TBC-R(5'-AACTACTGCTCAAACAAATAATGGA-3').Three-step method was used to amplify specific polymerase chain reaction(PCR)and the optimal PCR reaction system was determined by optimizing the PCR reaction procedure.At the same time,in order to ensure the accuracy of the test results,the PCR amplified products were conducted by generation sequencing.Results:The PCR method with the annealing temperature of 55-57℃and 37 cycles produced a single band at 237 bp for 21 batches of Eupolyphaga Steleophaga,20 batches of standard decoction and 19 batches of its dispensing granules,while it produced no band for the adulterant or the negative control.The experimental result was consistent with the result of Sanger sequencing,which was Eupolyphaga sinensis.Conclusion:The established specific PCR method can accurately identify the medicinal materials,and standard decoction freeze-dried powder of Eupolyphaga Steleophaga,as well as final products of dispensing granules.It provides a reference for research on the quality standards of Eupolyphaga Steleophaga dispensing granules.

关 键 词：土鳖虫(地鳖) 配方颗粒 细胞色素C氧化酶亚基1基因 混伪品 特异性聚合酶链式反应 分子鉴别 

分 类 号：R917[医药卫生—药物分析学]