摘要
目的利用COI序列对中药材蝉蜕Cicadae Periostracum及其混淆品进行DNA条形码鉴定研究,为蝉蜕药材的快速准确鉴定提供新的技术手段。方法收集全国市售蝉蜕药材、基地自采蝉蜕及其混淆品总45份样本,同时采集江苏徐州黑蚱养殖基地不同树种中蝉卵样品5份作为对照。提取DNA,进行PCR扩增及双向测序,测序结果采用DNAMAN和SnapGene进行拼接校对,运用MEGA11.0进行比对分析,计算种内及种间Kimura 2-Parameter(K2P)遗传距离并构建邻接法(neighbor-joining,NJ)系统发育树。结果黑蚱种内遗传距离远小于种间最小遗传距离,NJ树结果显示,黑蚱蝉蜕样品全部聚集为独立的一支,支持率为99%。混淆品样品分别聚集为单独的一支,支持率均大于90%,表明基于COI序列的DNA条形码技术可以有效鉴别蝉蜕药材真伪。结论应用COI序列作为DNA条形码能够准确有效地鉴别蝉蜕及其混淆品。
Objective To provide new technique for accurate identification of Chantui(Cicadae Periostracum)and its adulterants using DNA barcoding method based on the COI sequence.Methods Forty-five batches of the commercial Cicadae Periostracum and its adulterants,as well as self-harvested Cicadae Periostracum,were collected from different localities of China,and five batches of cicada eggs from different tree species in Xuzhou,Jiangsu Province were collected as controls.The total genomic DNAs of Cicadae Periostracum and its counterfeits were extracted.The COI fragments of these samples were amplified by PCR and sequenced in both directions to obtain the COI sequence.A total of 28 COI sequences of Cicadae Periostracum,17 adulterants and 9 related species were downloaded from Genbank database.The obtained sequences were assembled using the DNAMAN and SnapGene.MEGA11.0 was applied to calculate intraspecific and interspecific Kimura 2-Parameter(K2P)genetic distances and construct the neighbor-joining(NJ)phylogenetic tree.Results The intraspecific genetic distance of Heizha(Cryptotympana pustulata Fabricius)was much smaller than the minimum genetic distance between C.pustulata and its adulterants.The NJ tree showed that the genuine C.pustulata samples were all gathered as a separate one,with a support rate of 99%.The counterfeit samples were aggregated into another separate unit,and the support rate was greater than 90%,indicating that the DNA barcoding technology based on COI sequence could effectively identify the authenticity of Cicadae Periostracum.Conclusion COI sequence can stably and accurately distinguish Cicadae Periostracum and its adulterants,which provide a new technique to ensure the quality and clinical safety in utilization of Cicadae Periostracum.
作者
张欢欢
徐金娣
周静
贾聪玲
吴啟南
郭梦斐
沈纪晨
伍城颖
李松林
毛茜
ZHANG Huanhuan;XU Jindi;ZHOU Jing;JIA Congling;WU Qinan;GUO Mengfei;SHEN Jichen;WU Chengying;LI Songlin;MAO Qian(Department of Pharmaceutical Analysis,Affiliated Hospital of Integrated Traditional Chinese and Western Medicine,Nanjing University of Chinese Medicine,Nanjing 210028,China;Department of Metabolomics,Jiangsu Province Academy of Traditional Chinese Medicine,Nanjing 210028,China;School of Pharmacy,Nanjing University of Chinese Medicine,Nanjing 210028,China)
出处
《中草药》
CAS
CSCD
北大核心
2024年第6期2057-2065,共9页
Chinese Traditional and Herbal Drugs
基金
江苏省中医药科技发展计划重点项目(ZD202103)
江苏省中医药科技发展计划面上项目(MS202341)
南京中医药大学自然科学基金(XZR2021031)。
关键词
蝉蜕
混淆品
DNA条形码
COI序列
分子鉴定
Cicadae Periostracum
adulterants
DNA barcoding
COI sequence
molecular identification
作者简介
张欢欢(1999-),女,在读硕士研究生,研究方向为蝉蜕质量控制。E-mail:Zhanghuanhuan0404@163.com;通信作者:毛茜(1984-),女,博士,研究员,研究方向为中药药效物质基础和质量控制研究。E-mail:nancymq@126.com;通信作者:李松林(1964-),男,博士,研究员,研究方向为中药药效物质基础、质量控制和新产品研发。E-mail:songlinli64@126.com。
