中药材2025，Vol.48Issue(6)：1383-1388,6.DOI:10.13863/j.issn1001-4454.2025.06.008
特异性PCR方法鉴定大黄䗪虫丸中的水蛭成分
Authentication of Hirudo in Dahuang Zhechong Pills by Specific PCR Method
摘要
Abstract
Objective:To establish a specific PCR method for the identification of species authenticity of Hirudo in Dahuang Zhe-chong Pills,a Chinese patent medicine.Methods:Primers were designed based on the differences in the complete mitochondrial gene se-quences of Whitmania pigra(WP),Poecilobdella manillensis(PM),Hirudo nipponica(HN)and Whitmania laevis(WL).Total DNA was extracted and purified by cetyltrimethylammonium bromide(CTAB)and phenol mimesis.Based on this template,polymerase chain reaction(PCR)was established and the reaction conditions were screened and optimized.The specificity and sensitivity of the method were investigated,and the Hirudo components in commercially available Dahuang Zhechong Pills were measured.Results:The four pairs of specific primers PWP,PHN,PPM and PWL obtained from the design amplified clear target bands at 63,154,155 and 197 bp,re-spectively,and no interference from non-specific bands was observed.The detection limits of the four pairs of specific primers were all 1.0 ng/μL.Feasibility verification results showed that three out of ten commercially available batches of Dahuang Zhechong Pills were identified as qualified products with only DNA of Whitmania pigra.The target bands were not detected in the remaining 7 batches.Con-clusion:The method established in this study has good specificity and sensitivity,which can be used to identify Hirudo in the classic prescription Dahuang Zhechong Pills,providing a new detection method for the market regulation and origin tracing of Hirudo-containing formulations.关键词
大黄䗪虫丸/水蛭/特异性引物/PCR扩增Key words
Dahuang Zhechong Pills/Hirudo/Specific primers/PCR amplification分类
医药卫生引用本文复制引用
WU Zhen-hua,XU Chao,WANG Jia-qi,LU Xiao-ling,XIA Guo-hua,YANG Chun-bin,YANG Huan..特异性PCR方法鉴定大黄䗪虫丸中的水蛭成分[J].中药材,2025,48(6):1383-1388,6.基金项目
国家自然科学基金项目(82173937) （82173937）
镇江市科技计划项目(SH2023014) （SH2023014）
江苏大学大学生创新创业训练计划项目(202410299966X) （202410299966X）
