特异性PCR方法鉴定大黄䗪虫丸中的水蛭成分OA北大核心

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.