应用聚合酶链式反应技术快速鉴别中药水蛭

Rapid Identification of Hirudo by Species-specific Polymerase Chain Reaction Technology

目的 建立聚合酶链式反应法(polymerase chain reaction,PCR)对中药水蛭进行快速种间鉴别。方法 根据中国药典项下3种水蛭(宽体金线蛭、日本医蛭及尖细金线蛭)CO1序列的差异设计并合成3对种属特异性引物,对其进行PCR反应条件优化;对不同批次的水蛭进行稳定性试验,并对同批次的水蛭进行灵敏度试验;利用优化的PCR条件对水蛭混合样品进行分析。结果 3对引物分别能在60,61,59℃的退火温度下扩增出1 000~2 000,1 000~2 000,3 000 bp左右的明亮条带,检测限分别为0.1,1,10 ng,并能够准确鉴别以不同比例混合的水蛭样品。结论 所设计的3对引物可以快速鉴别中药水蛭、常见伪品及混掺水蛭,并具有较高的种间特异性及良好的种内稳定性和灵敏度,可作为中药水蛭传统基原鉴别方法的补充,为其质量控制提供更多参考。

OBJECTIVE To establish a polymerase chain reaction(PCR) method for rapid interspecific identification of Hirudo.METHODS Three pairs of specie-specific primers were designed and synthesized according to the differences of CO1 sequences of three kinds of Hirudo(Whitmania pigra Whitman,Hirudo nipponica Whitman,Whitmania acranulata Whitman) in the Chinese Pharmacopoeia,and the PCR reaction conditions were optimized.The stability experiment was carried out for different batches of Hirudo,and the sensitivity experiment was carried out for the same batch of Hirudo.The optimized PCR conditions were used to analyze the mixed Hirudo samples.RESULTS The 3 pairs of primers could amplify the bright bands of 1 000–2 000,1 000–2 000 and 3 000 bp at annealing temperature of 60,61 and 59 ℃ respectively,with detection limit of 0.1,1 and 10 ng respectively,and could accurately identify the Hirudo samples mixed in different proportions.CONCLUSION The 3 pairs of primers can be used for the rapid identification of Hirudo,common counterfeit and mixed Hirudo with high interspecific specificity and good intra-specific stability and sensitivity,which can be used as a supplement to the traditional identification method of Hirudo and provide more reference for its quality control.