特异性PCR鉴别蜈蚣及其混伪品
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摘要
目的:蜈蚣是我国传统的中药材,具有良好的药用价值。目前市场上蜈蚣的混伪品日益增多,临床用药的安全性和有效性难以保证。该研究建立了一种蜈蚣基原物种的特异性聚合酶链式反应(PCR)方法,以准确、简便的鉴别蜈蚣及其常见混伪品。方法:通过比较蜈蚣及其混伪品基原物种的COI基因序列差异,根据变异位点设计蜈蚣正品少棘巨蜈蚣的特异性鉴别引物WG-500.F和WG-500.R,采集蜈蚣原动物样本及药材样本,优化反应体系并对此方法进行耐受性和适用性的考察和验证。结果:少棘巨蜈蚣原动物样本及蜈蚣正品药材使用设计的蜈蚣鉴别引物经PCR扩增和凝胶电泳后出约500 bp的单一明亮条带,其他混伪品如多棘蜈蚣、模棘蜈蚣、哈氏蜈蚣、海南蜈蚣等均无条带出现。结论:PCR鉴别方法可准确鉴别蜈蚣原动物和药材的真伪,具有高度特异性,为中药材蜈蚣的真伪鉴别提供了良好的科学依据。该方法具有简单、直观等特点,有利于广泛的普及与应用,在中药材鉴定方面有着广阔的应用前景。
Objective:Scolopendra was a traditional Chinese medicine(TCM) with a good medicinal value.Nowadays,there have been increasingly more adulterates of Scolopendra in the medicine market.To ensure the safe and effectiveness of clinical medicines,a convenient and accurate specific polymerase chain reaction(PCR) method for identification of medicinal Scolopendra from its common adulterates was established.Method:Based on the differences of COI gene DNA sequences among Scolopendra subspinipes mutilans and adulterants,the specific primer was designed,the reaction conditions were optimized,and the PCR method for identification was explored and verified in terms of tolerance and feasibility.Besides,the original animal samples and medicine of Scolopendra were collected.Result:Through the established PCR reaction system,the bright and simple fragments of 500 bp was amplified from DNA templates of S.subspinipes mutilans.All of the adulterants were negative by the multiplex PCR assay,such as S.multidens,S.subspinipes,S.dehaani,S.hainanum.Conclusion:The identified primer is highly specific,and the specific PCR method established in this paper can accurately identify Scolopendra and its adulterants,so as to provide an excellent scientific basis for the identification of TCM Scolopendra.The method is simple and intuitive,and facilitates wide promotion and application of the method,with a broad application prospect in the identification of TCM.
Objective:Scolopendra was a traditional Chinese medicine(TCM) with a good medicinal value.Nowadays,there have been increasingly more adulterates of Scolopendra in the medicine market.To ensure the safe and effectiveness of clinical medicines,a convenient and accurate specific polymerase chain reaction(PCR) method for identification of medicinal Scolopendra from its common adulterates was established.Method:Based on the differences of COI gene DNA sequences among Scolopendra subspinipes mutilans and adulterants,the specific primer was designed,the reaction conditions were optimized,and the PCR method for identification was explored and verified in terms of tolerance and feasibility.Besides,the original animal samples and medicine of Scolopendra were collected.Result:Through the established PCR reaction system,the bright and simple fragments of 500 bp was amplified from DNA templates of S.subspinipes mutilans.All of the adulterants were negative by the multiplex PCR assay,such as S.multidens,S.subspinipes,S.dehaani,S.hainanum.Conclusion:The identified primer is highly specific,and the specific PCR method established in this paper can accurately identify Scolopendra and its adulterants,so as to provide an excellent scientific basis for the identification of TCM Scolopendra.The method is simple and intuitive,and facilitates wide promotion and application of the method,with a broad application prospect in the identification of TCM.
引文
[1]国家药典委员会.中华人民共和国药典.一部[M].北京:中国医药科技出版社,2015:122.
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[3]王勇炫,吴伟康,吴以岭,等.蜈蚣有效部位抗心肌缺血作用及其NO机制研究[C]//络病学基础与临床研究(6),2010-09,天津:中华中医药学会络病分会,2010:313-318.
[4]汪梅姣,谢志军,谷焕鹏,等.蜈蚣、地龙、地鳖虫镇痛作用比较的实验研究[J].中国中医急症,2012,21(9):1435-1436.
[5] Siriwut W,Edgecombe G D,Sutcharit C,et al. A taxonomic review of the centipede genus Scolopendra Linnaeus,1758(Scolopendromorpha,Scolopendridae)in mainland Southeast Asia,with description of a new species from Laos[J]. Zoo Keys,2016(590):1-66.
[6]康四和.中国蜈蚣属动物分类鉴定及药用蜈蚣药材质量评价研究[D].武汉:湖北中医药大学,2018.
[7]宋志顺.中国蜈蚣目的分类研究(多足总纲:唇足纲)[D].保定:河北大学,2004.
[8]蒋超,金艳,袁媛,等.羚羊角塞特异性PCR鉴别方法研究[J].世界中医药,2018,13(2):252-255.
[9]刘富艳,金艳,袁媛,等.多重位点特异性PCR鉴别海龙及其混伪品[J].中国实验方剂学杂志,2018,24(15):57-64.
[10]何志一,唐先明,刘建辉,等.快速PCR方法在哈蟆油真伪鉴别中的应用研究[J].中国中药杂志,2017,42(13):2467-2472.
[11]蒋超,赵群,金艳,等.快速PCR技术鉴别中药材蛤蚧的方法研究[J].中国现代中药,2017,19(1):21-25.
[12]陈康,蒋超,袁媛,等.快速PCR方法在蛇类药材真伪鉴别中的应用[J].中国中药杂志,2014,39(19):3673-3677.
[13] Kronmüller C. Review of the subspecies of Scolopendra subspinipes Leach,1815 with the new description of the South Chinese member of the genus Scolopendra Linnaeus,1758 named Scolopendra hainanum spec. Nov[J]. Spixiana,2012,35(1):19-28.
[14]王克勤,方红,叶卯祥,等.蜈蚣药源调查及商品鉴定[J].中药材,1997,20(9):450-452.
[15]胡云飞,鞠康,荣百玲,等.不同品种进口蜈蚣的微性状鉴别[J].中国实验方剂学杂志,2017,23(8):39-44.
[2]王硕,覃文慧.多棘蜈蚣与少棘蜈蚣抗肿瘤作用研究[J].中国实验方剂学杂志,2011,17(13):156-158.
[3]王勇炫,吴伟康,吴以岭,等.蜈蚣有效部位抗心肌缺血作用及其NO机制研究[C]//络病学基础与临床研究(6),2010-09,天津:中华中医药学会络病分会,2010:313-318.
[4]汪梅姣,谢志军,谷焕鹏,等.蜈蚣、地龙、地鳖虫镇痛作用比较的实验研究[J].中国中医急症,2012,21(9):1435-1436.
[5] Siriwut W,Edgecombe G D,Sutcharit C,et al. A taxonomic review of the centipede genus Scolopendra Linnaeus,1758(Scolopendromorpha,Scolopendridae)in mainland Southeast Asia,with description of a new species from Laos[J]. Zoo Keys,2016(590):1-66.
[6]康四和.中国蜈蚣属动物分类鉴定及药用蜈蚣药材质量评价研究[D].武汉:湖北中医药大学,2018.
[7]宋志顺.中国蜈蚣目的分类研究(多足总纲:唇足纲)[D].保定:河北大学,2004.
[8]蒋超,金艳,袁媛,等.羚羊角塞特异性PCR鉴别方法研究[J].世界中医药,2018,13(2):252-255.
[9]刘富艳,金艳,袁媛,等.多重位点特异性PCR鉴别海龙及其混伪品[J].中国实验方剂学杂志,2018,24(15):57-64.
[10]何志一,唐先明,刘建辉,等.快速PCR方法在哈蟆油真伪鉴别中的应用研究[J].中国中药杂志,2017,42(13):2467-2472.
[11]蒋超,赵群,金艳,等.快速PCR技术鉴别中药材蛤蚧的方法研究[J].中国现代中药,2017,19(1):21-25.
[12]陈康,蒋超,袁媛,等.快速PCR方法在蛇类药材真伪鉴别中的应用[J].中国中药杂志,2014,39(19):3673-3677.
[13] Kronmüller C. Review of the subspecies of Scolopendra subspinipes Leach,1815 with the new description of the South Chinese member of the genus Scolopendra Linnaeus,1758 named Scolopendra hainanum spec. Nov[J]. Spixiana,2012,35(1):19-28.
[14]王克勤,方红,叶卯祥,等.蜈蚣药源调查及商品鉴定[J].中药材,1997,20(9):450-452.
[15]胡云飞,鞠康,荣百玲,等.不同品种进口蜈蚣的微性状鉴别[J].中国实验方剂学杂志,2017,23(8):39-44.