摘要
目的:近年来,随着半夏和天南星的野生资源急剧下降、药材栽培技术不成熟等问题出现,市场的混伪品频繁出现,而其主要鉴别特征在药材流通中大多消失。因此,迫切需要建立一种可以快速、有效鉴别天南星和半夏特异性的分子鉴定方法。方法:通过比对天南星、半夏及其混伪品的rbc L序列,分别选择了天南星和半夏的特异性酶切位点HaeⅢ和DraⅠ,采用聚合酶链式反应-限制性片段长度多态性(PCR-RFLP)进行鉴定,建立并优化了PCR-RFLP反应主要体系条件,并对其耐用性和正品、掺杂品及混伪品的检出能力进行了考察。结果:建立了天南星和半夏药材的PCR-RFLP鉴别方法,在退火温度为54℃、循环次数为35个,DNA模板量为3~30 ng时,天南星和半夏经过特异性引物扩增后,分别能被HaeⅢ和DraⅠ限制性内切酶酶切,产生两片段或切开的小片段,并在100~250 bp出现单一条带,而混伪品无法酶切,且均在250 bp出现条带,该方法对天南星或半夏的掺杂品和混伪品具有高度准确的鉴别能力。结论:该研究建立的PCR-RFLP方法可以快速鉴别天南星、半夏。
Objective:In recent years,with the sharp decline of wild resources in Arisaematis Rhizoma and Pinelliae Rhizoma and the immaturity of medicinal cultivation technology,their adulterants have appeared frequently in the market,and the main identifying characteristics have mostly disappeared in the circulation of medicinal materials.Therefore,there is an urgent need to establish a molecular identification method that can quickly and effectively identify the specificity of Arisaematis Rhizoma and Pinelliae Rhizoma.Method:After comparison of the rbc L sequences of Arisaematis Rhizoma,Pinelliae Rhizoma,and their adulterants,the specific enzyme cleavage sites HaeⅢand DraⅠof Arisaematis Rhizoma and Pinelliae Rhizoma,respectively,were selected and identified by polymerase chain reaction-restriction fragment length polymorphism(PCRRFLP).The main system conditions of PCR-RFLP reaction were established and optimized,and their durability and the ability to detect genuine,adulterants,and mixed counterfeits were investigated.Result:The PCRRFLP identification method of Arisaematis Rhizoma and Pinelliae Rhizoma was established.After specific primer amplification,Arisaematis Rhizoma and Pinelliae Rhizoma could be digested by HaeⅢand DraⅠ-restricted endonucleases respectively,at annealing temperature of 54℃,the number of cycles of 35,and the amount of DNA template of 3-30 ng,producing two fragments or small cut fragments with a single band between 100-250 bp,whereas the mixed counterfeits were not cleaved and both showed a band at 250 bp.The method is highly accurate in identifying adulterants and mixed counterfeits of Arisaematis Rhizoma or Pinelliae Rhizoma.Conclusion:The PCR-RFLP method developed in this study allows for the rapid identification of Arisaematis Rhizoma and Pinelliae Rhizoma.
作者
肖建才
闫滨滨
杨健
孙嘉惠
王铁霖
万修福
孙楷
蒋超
张燕
XIAO Jiancai;YAN Binbin;YANG Jian;SUN Jiahui;WANG Tielin;WAN Xiufu;SUN Kai;JIANG Chao;ZHANG Yan(State Key Laboratory and Breeding Base of Dao-di Herbs,Institute of Chinese Materia Medica,China Academy of Chinese Medical Sciences,Beijing 100700,China)
出处
《中国实验方剂学杂志》
CAS
CSCD
北大核心
2023年第6期194-201,共8页
Chinese Journal of Experimental Traditional Medical Formulae
基金
国家自然科学基金项目(81891014)
中国中医科学院研究生院教材和教改项目(KCSZ21001)。
关键词
天南星
半夏
聚合酶链反应-限制性片段长度多态性方法
混伪品
分子鉴定
Arisaematis Rhizoma
Pinelliae Rhizoma
polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP)
adulterants
molecular identification
