地龙的多重位点特异性PCR鉴别

Identification of Pheretima by Multiplex Allele-specific PCR

目的:近年来,中药材地龙的商品价格不断上涨,市场的混伪品随之增加。其主要鉴别特征在药材中大部分丢失,难以区分。因此,急需建立一种准确、稳定的中药材地龙基原鉴别方法。方法:根据地龙及其混伪品的COI基因序列差异找到变异位点,从而设计参环毛蚓、通俗环毛蚓、威廉环毛蚓及栉盲环毛蚓的特异性鉴别引物,优化反应体系并进行耐受性和适用性的考察和验证。在此基础上将特异性引物组合,构建多重聚合酶链式反应(PCR)体系,从而建立了一种高效准确、操作简便、专属性强、重复性好的中药材地龙及其常见混伪品的多重位点PCR鉴别方法。结果:在该方法下广地龙基原参环毛蚓可得到366 bp片段,沪地龙基原通俗环毛蚓和威廉环毛蚓可得到487 bp片段,栉盲环毛蚓可得到475 bp片段,其他混伪品均无条带。将之组合形成的多重PCR可在一次鉴定操作中根据条带位置同时鉴别沪地龙和广地龙。结论:该文所建立的多重位点特异性PCR鉴别方法可准确鉴别中药材地龙真伪。

Objective:In recent years,with the increase in the commodity price of medicinal pheretima,there have emerged increasing adulterates in the medicine market.Besides,the medicinal materials have mostly lost the main identification features,and are difficult to distinguish.Therefore,it is urgent to establish an accurate and stable method for the identification of pheretima.Method:According to the differences of COI gene DNA sequences among Pheretima aspergillum,Pheretima vulgaris,Pheretima guillelmi,Pheretima pectinifera and adulterants,the variation site was found,the specific primers were designed,the reaction conditions were optimized,and the polymerase Chain reaction(PCR)method for identification was explored and verified in terms of tolerance and feasibility in this study.The specific primers were combined to build multiple PCR systems.An effective,accurate,convenient,highly specific and repeatable Multiplex Allele-Specific PCR identification method was established for identifying medicinal pheretima and its common adulterants.Result:Through the established multiplex PCR reaction system,366,487,487 and 475 bp of fragments were amplified from DNA templates of P.aspergillum,P.vulgaris,P.guillelmi and P.pectinifera respectively.All of the adulterants were negative by the multiplex PCR assay.The PCR amplification of specific alleles method established in this paper can accurately identify pheretima.Conclusion:The Multiplex Allele-Specific PCR identification method established in this paper can accurately identify medicinal pheretima and its adulterants.