脑心通胶囊中水蛭的特异性DNA鉴别

Identification of specific DNA markers for Hirudo in the Naoxintong capsule

建立正品水蛭(宽体金线蛭、日本医蛭)的DNA分子鉴别方法。基于线粒体细胞色素c氧化酶亚基Ⅰ(COⅠ)基因序列,分别设计了针对宽体金线蛭和日本医蛭的特异性引物,将其应用于水蛭药材正品、常见伪品菲牛蛭以及含水蛭的中成药脑心通胶囊的PCR技术检测。结果显示单独使用引物WF1R2或WF2R2均可以特异性扩增宽体金线蛭的DNA,产生大小为200 bp左右的条带;而引物HF1R2则可以特异性扩增日本医蛭,产生大小为142 bp的条带;这3对引物在伪品菲牛蛭中均无扩增条带。且用引物WF1R2和WF2R2均在脑心通胶囊中检测到宽体金线蛭,与胶囊所用投料药材一致。结果表明这3对引物可以分别对宽体金线蛭和日本医蛭进行特异性鉴别,且无需测序;而引物WF1R2和WF2R2可直接用于脑心通胶囊中所用水蛭正品的检控。该DNA分子鉴定方法简便、准确,特异性强且灵敏度高,可作为对传统基原鉴定方法的补充,进一步提高水蛭药材及含水蛭的中成药脑心通胶囊的质量控制水平。

To identify the genuine species of Hirudo(Whitmania pigra,Hirudo nipponica),DNA molecular method was established.Based on mitochondrial cytochrome c oxidase subunitⅠ(COⅠ)gene,species-specific primers were designed for W.pigra and H.nipponica,respectively.Using three species-specific primers,PCR amplification was performed for the genuine species and the common adulterant Poecilobdella manillensis Lesson,as well as the Naoxintong capsules.Results showed that both primers WF1R2 and WF2R2 could specifically amplify the DNA of W.pigra with amplicons of around 200 bp,while the primers HF1R2 could specifically amplify the DNA of H.nipponica to generate amplicons of 142 bp.Besides,no amplification was obtained in the adulterant P.manillensis with above three pairs of primers.In addition,amplicons around 200 bp were obtained in Naoxintong capsules using primers WF1R2 or WF2R2,with the same sequences as W.pigra,which is the genuine species that the company used.These results indicated that without sequencing,the primers WF1R2(or WF2R2)and HF1R2 could specifically amplify W.pigra and H.nipponica,respectively.Furthermore,the primers WF1R2 and WF2R2 could be applied to identify the genuine species in Naoxintong capsule.This DNA molecular identification method is simple,accurate,with high specificity and sensitivity,thus it could be used as a supplement to the conventional method for origin identification and further improve the quality control of Hirudo and the Hirudo in Naoxintong capsule.