摘要
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.
作者
田娜
魏艺聪
袁媛
金艳
杨全
赵玉洋
蒋超
TIAN Na;WEI Yi-cong;YUAN Yuan;JIN Yan;YANG Quan;ZHAO Yu-yang;JIANG Chao(College of Traditional Chinese Medicine,Guangdong Pharmaceutical University,Guangzhou 510006,China;State Key Laboratory Breeding Base of Dao-di Herbs,China Academy of Chinese Medical Sciences,National Resource Center for Chinese Materia Medica,Beijing 100700,China)
出处
《中国实验方剂学杂志》
CAS
CSCD
北大核心
2019年第17期124-129,共6页
Chinese Journal of Experimental Traditional Medical Formulae
基金
中央级公益性科研院所基本科研业务费专项(ZZ10-008)
中央本级重大增减支项目(2060302)
中国中医科学院中药资源中心自主选题项目(ZZXT201709)
关键词
地龙
多重位点特异性聚合酶链式反应
分子鉴别
混伪品
参环毛蚓
通俗环毛蚓
威廉环毛蚓
栉盲环毛蚓
Pheretima
multiplex allele-specific polymerase chain reaction
molecular identification
adulterants
Pheretima aspergillum
Pheretima vulgaris
Pheretima guillelmi
Pheretima pectinifera