动物药材DNA提取方法优化和市售药材DNA条形码鉴定研究  

DNA Extraction Method Optimization for Animal Medicines and Identification of Commercial Animal Medicines Using DNA Barcoding

作　　者：刘旭朝[1,2] 刘金欣[2,3] 孙伟[4] 宋经元[2] 石林春[2] 孙稚颖[1] Liu Xuzhao Liu Jinxin Sun Wei Song Jingyuan Shi Linchun Sun Zhiying(School of Pharmacy, Shandong University of Traditional Chinese Medicine, Jinan 250355, China Institute of Medicinal Plant Development /Key Laboratory of Bioactive Substances and Resources Utilization of Chinese Herbal Medicine, Ministry of Education, Chinese Academy of Medical Sciences ＆ Peking Union Medical College, Beijing 100193, China Chengde Medical University/Hebei Key Laboratory of Research and Development for Traditional Chinese Medicine, Chengde 067000, China Institute of Chinese Materia Medica/Key Laboratory of Beijing for Identification and Safety Evaluation of Chinese Medicine, China Academy of Chinese Medical Sciences, Beijing 100700, China)

机构地区：[1]山东中医药大学药学院,济南250355 [2]中国医学科学院北京协和医学院药用植物研究所/中草药物质基础与资源利用教育部重点实验室,北京100193 [3]承德医学院/河北省中药研究与开发重点实验室,承德067000 [4]中国中医科学院中药研究所/中药鉴定与安全性检测评估北京市重点实验室,北京100700

出　　处：《世界科学技术-中医药现代化》2017年第4期602-609,共8页Modernization of Traditional Chinese Medicine and Materia Medica-World Science and Technology

基　　金：国家中医药管理局公益性行业科研专项经费项目分任务(201507002-4-1-2):海龙等4种名贵珍稀动物药及混伪品鉴定技术及应用规范;负责人:张辉;中国医学科学院医学与健康科技创新工程本草基因组协同创新团队(2016-I 2M-3-016):负责人:宋经元

摘　　要：目的:对动物药材DNA提取方法进行优化,利用优化方法提取市售动物药材DNA并进行DNA条形码鉴定。方法:基于SDS法DNA提取原理,比较裂解液中不同EDTA浓度(0.025、0.25、0.5 mol·L^(-1))、是否含Na Cl和Triton X-100等因素对不同用药部位动物药材DNA提取质量的影响,筛选得到最佳裂解液配方;使用优化的裂解液配方提取121份市售动物药材DNA并进行基原物种鉴定。结果:裂解液配方为1%SDS、0.03 mol·L^(-1)Tris-HCl、0.25 mol·L^(-1)EDTA、0.2 mol·L^(-1)Na Cl对不同用药部位动物药材DNA提取效果最佳,并可实现对蝉蜕等提取困难样本DNA的提取;利用优化裂解液提取的121份市售动物药材DNA满足中药材分子鉴定后续实验要求,所有市售动物药材均可准确鉴定到基原物种。结论:本研究优化的裂解液配方可用于除壳类、分泌物类、加工品外不同用药部位动物药材的DNA提取,为动物药材分子鉴定提供了技术支持。The DNA extraction method of animal medicine material is difficult and un-unified, which limits the application of molecular identification to identify animal medicines. In this study, based on the DNA extraction theory of SDS, we assessed the effects of three elements including different EDTA concentrations （0.025 mol·L^-1, 0.25 mol·L^-1, and 0.5 mol·L^-1） and whether containing NaC1 and Triton X-100 in the lysis buffer on the quality of DNA extracted from different kinds of animal medicine. The optimized lysis buffer was used to extract DNA from 121 commercial animal medicines for original and species identification. The results showed that the lysis buffer of 1% SDS, 0.03 mol·L^-1 Tris- HCl, 0.25mol·L^-1 EDTA and 0.2mol·L^-1 NaCl had the optimum effect on DNA extraction. This lysis buffer can obtain DNA from animal medicine which is difficult to extract, such as Cicadae periostracum. The DNA extractions of 121 commercial animal medicines by optimized lysis buffer can satisfy the experimental requirements for molecular identification. All samples of commercial animal medicines can be accurately identified to the level of species. It was concluded that optimized lysis buffer can be used in the DNA extraction of different kinds of animal medicines except shells, secretions and processed products. This method provides technique support for the molecular identification of animal medicines.

关 键 词：动物药材 DNA条形码 DNA提取 裂解液 EDTA 

分 类 号：R931.2[医药卫生—生药学]