宽叶山蒿与艾饮片及其叶绒的特异性PCR鉴别  

Distinguishing between Artemisia stolonifera and A.argyi by specific PCR of leaves and non-glandular trichomes

作　　者：赵亚臣 李双鸽 李慧[3,4] 刘义梅 赵婷婷[2] 苗玉焕 刘大会 黄璐琦[5] ZHAO Ya-chen;LI Shuang-ge;LI Hui;LIU Yi-mei;ZHAO Ting-ting;MIAO Yu-huan;LIU Da-hui;HUANG Lu-qi(Aademician Workstation,Jiangxi University of Chinese Medicine,Nanchang 330004,China;Resource Center for Chinese Materia Medica,Hubei University of Chinese Medicine,Wuhan 430065,China;Institute of Traditional Chinese Medicine Health Industry,China Academy of Chinese Medical Sciences,Nanchang 330000,China;Jiangxi Health Industry Institute of Traditional Chinese Medicine,Nanchang 330000,China;China Academy of Chinese Medical Sciences,Beijing 100700,China)

机构地区：[1]江西中医药大学院士工作站,江西南昌330004 [2]湖北中医药大学中药资源中心,湖北武汉430065 [3]中国中医科学院,中医药健康产业研究所,江西南昌330000 [4]江西中医药健康产业研究院,江西南昌330000 [5]中国中医科学院,北京100700

出　　处：《中国中药杂志》2023年第14期3730-3735,共6页China Journal of Chinese Materia Medica

基　　金：江西中医药健康产业研究院科技项目;国家中医药管理局青年岐黄学者项目;湖北省现代农业产业技术体系项目(HBHZD-ZB-2020-005);湖北省教育厅中青年人才项目(Q20212001)。

摘　　要：宽叶山蒿为艾的近源种,两者饮片或叶绒混在一起很难鉴别。该研究旨在从分子层面建立一种快速鉴别宽叶山蒿与艾的方法。根据宽叶山蒿与艾的ITS2基因测序结果,使用DNAMAN软件进行序列比对,分析得到在ITS2基因202位存在C/T单核苷酸多态性位点(single nucleotide polymorphism,SNP);并依据此SNP位点设计特异性引物,建立并优化特异性PCR方法,以此方法进行混样检测验证可行性与稳定性。在特异性PCR条件下,使用特异性引物JNC-F与通用引物ITS3R,艾可以扩增出1条218 bp的特异条带,而宽叶山蒿不能扩增;使用特异性引物KY-F与通用引物ITS3R,宽叶山蒿可以扩增出218 bp特异条带,而艾不能扩增。建立的方法同时可以用于检测饮片及加工品叶绒,使用JNC-F/ITS3R体系可以检测宽叶山蒿中是否混有艾,艾饮片及叶绒的最低检测限均为3%;使用KY-F/ITS3R体系可以检测艾中是否混有宽叶山蒿,宽叶山蒿饮片及叶绒的最低检测限均为5%。模板检出限考察中2个体系对模板的检出限均为5 ng。构建方法可为两者质量控制提供依据,准确检测和判定市场上2种药材是否混用。Artemisia stolonifera is a relative of A.argyi.The two species are difficult to be distinguished due to the similarity in leaf shape and have even less distinctive features after processing.This study aims to establish a method to quickly distinguish between them.At the same time,we examined the reasonability and applicability of the specific polymerase chain reaction(PCR)method.The C/T single nucleotide polymorphism was detected at the position 202 of the sequence,based on which specific primers were designed to identify these two species.The PCR with the specific primer JNC-F and the universal primer ITS3R produced a specific band at 218 bp for A.argyi and no band for A.stolonifera,which can be used to detect at least 3%of A.argyi samples mixed in A.stolonifera samples.The PCR with the specific primer KY-F and the universal primer ITS3R produced a specific band at 218 bp for A.stolonifera and no band for A.argyi,which can be used to detect at least 5%of A.stolonifera samples mixed with A.argyi.The limit of detection of the established method was 5 ng DNA.The established PCR method can accurately distinguish between A.stolonifera and A.argyi,which provides an experimental basis for the quality control of A.stolonifera and determines whether the herbs are adulterated.

关 键 词：宽叶山蒿 艾 叶绒 ITS2 单核苷酸多态性位点 分子鉴定 

分 类 号：R282.5[医药卫生—中药学]