中药鉴定创新方法体系的建立与推广 (2).ppt

中药鉴定创新方法体系的建立与推广 DNA barcoding system for herbal materials,陈士林 Shilin Chen 中国中医科学院中药研究所,中药鉴定技术瓶颈,一万余种中药材物种鉴定 品种混乱 传统四大鉴定方法体系的局限 路在何方？ 基原鉴定 形态鉴定 显微鉴定 理化鉴定,DNA条形码 中药鉴定领域的变革与突破,经典中药鉴定方法难以满足现代需求 鉴定方法缺失导致严重安全性事件 - 关木通误当木通服用，导致急性肾功能衰竭（马兜铃酸肾病事件） -土三七误当三七服用，导致严重肝毒性,Hoang et al. Science Translational Medicine, 2013, 5: 197ra102. Nortier J L, Martinez M C M, Schmeiser H H, et al. New England Journal of Medicine, 2000, 342(23): 1686-1692.,关木通 百度百科,木通 中药材天地网,DNA barcoding can achieve a rapid and accurate identification for herbs,DNA barcoding technology uses a short and standardized gene region to identify species,One sequence One species,课题组首次提出并验证ITS2条形码，引起国内外广泛关注,国际期刊PLoS ONE关注药用植物DNA条形码。该文提出ITS2序列为药用植物通用的条形码序列，为陆地植物DNA条形码序列筛选提出了新的视角，由此引起同行的关注和讨论。,该文发表后3年引文288次,首次验证 ITS2为药用植物鉴定通用条形码,通过研究比较psbA-trnH, matK, rbcL, rpoC1, ycf5, ITS2 和 ITS等七个候选DNA条形码，发现ITS2是最佳条形码序列 ITS2长度约230bp，易于扩增和测序，物种鉴定能力强，适合中药材等存在DNA降解材料的鉴定 人参、西洋参、羌活、宽叶羌活DNA条形码鉴定方法正在纳入国家药典,Relative distribution of inter-specific divergence between congeneric species and intra-specific variation for four loci. The two bars in each box represent inter-specific (above) and intra-specific (below) genetic distances.,Identification of the family Rosaceae,Wilcoxon signed-rank tests of inter-specific divergence among loci.,ITS2 and rbcL, matK, rpoC1 were compared. For 54 samples from 43 species in 19 diverse genera, the successful identification rate with ITS2 was 100%, followed by matK (94%) and rbcL(65%). For 1410 samples from 893 species in 96 genera No. of genera (2 species): 47 (1351 samples) No. of species ( 2 samples ): 297 (814 samples) Identification rate with ITS2 was 100% and 78% at genus and species level, respectively.,X Pang, Shilin Chen*. Cladistics, 2011, 27,Comparative analysis of a large dataset indicates that ITS should be incorporated into the core barcode for seed plants China Plant BOL Group, Li Dezhu et al., PNAS, 2011,We therefore propose that ITS/ITS2 should be incorporated into the core barcode for seed plants. (建议ITS/ITS2应成为种子植物的核心条形码) In cases where it is difficult to amplify and directly sequence ITS in its entirety, just using ITS2 is a useful back-up as it is easier to amplify and sequence this subset of the marker. (ITS难以扩增和测序时，ITS2可以有效地弥补该缺陷),创建ITS2为主体条形码的中草药鉴定体系,研究一万余份药材样本（涉及数千个基原物种） 70余篇相关文章发表于Cladistics、PLoS ONE、PNAS、BMC Evol Biol、 中国科学等杂志 出版中药DNA条形码分子鉴定 人民卫生出版社 起草中国药典中药材DNA条形码分子鉴定指导原则，已获准纳入国家药典 建立中药DNA条形码分子鉴定标准流程,冬虫夏草及其混伪品/近缘种,背景：分布范围狭窄，生态环境脆弱，资源蕴藏量有限（产量每年80-100吨）； 市场产不足销，价格已超黄金 (Guo, 2012) 混伪品泛滥，如古尼虫草，蛹虫草，人造伪品滇紫菀等 样品：冬虫夏草与13种混伪品及近缘种，冬虫夏草采自四川、青海、西藏等主产区 185 条ITS/ITS2序列 (冬虫夏草样品53份，伪品45份，GenBank序列87 条),A：正品：冬虫夏草 B：伪品： 亚香棒虫草/古尼虫草 C：人造伪品： 滇紫菀 D：伪品 凉山虫草 E：伪品：蝉花 F：伪品：下垂虫草 G：伪品： Cordyceps robertsii（新西兰）,结果：基于BLAST/DISTANCE的鉴定效率：100% 基于NJ tree 的鉴定结果：所有的冬虫夏草序列单独聚为一支， 并能与所有的混伪品及近缘种区分开,Xiang L, Chen SL, et al. FEMS microbiology letters, 2013, 347(2): 156-162.,DNA条形码鉴定冬虫夏草及其伪品,中国药典中药材DNA条形码分子鉴定指导原则,Morphological characteristics of the powder, decoction pieces, seed ,seedling, and fibre among the P.ginseng , P. quinquefolius, P notoginseng and its closely related species are similar. It is difficult to identify so many different panax species and ginseng produce in medicinal uses by traditional classification methods.,Using DNA barcode to identify panax medicinal species 人参类及其混伪品的鉴定,13,1 5 10 15 20 25 30 35 40 45 50,SN01: p.ginseng;SN03: p.quinquefolius,显示人参、西洋参ITS2序列的SNPs位点信息。Position 32 and 43为区别人参和西洋参的SNPs位点 为验证该方法准确性，我们下载了GenBank所有人参、西洋参ITS2序列，表明两个SNPs位点稳定存在,样品来源：采集了主产区吉林，辽宁，黑龙江，北京，山东,加拿大及上海和香港药市人参西洋参样品共计72份,ITS2 sequence position 1-50 of p.ginseng and p. quinquefolius,方 法,结 果,结 论,基于ITS2条形码鉴定人参和西洋参,Gene, 2013, 530(1): 39-43.,A Fast SNP Identification of Medicinal Panax Species Based on DNA Barcoding Analysis,14,金银花药材及其混伪品的ITS2条形码鉴定,研究对象：包括24份金银花药材。,M: marker 1、26: 阴性对照(CK) 2-25: 金银花 27-47：混伪品,DNA提取： 取金银花药材约25 mg，利用植物基因组DNA提取试剂盒(Tiangen Biotech Co., China)提取总DNA。 ITS2序列PCR扩增程序及电泳图,BLAST鉴定,Identification of Cortex Herbs,Sun ZY, Chen SL*. J Nat Med. 2012.,The identification efficiency of 51 samples belonging to 19 kinds of cortex herbs in Chinese Pharmacopoeia examined was 100% using ITS2 sequence as DNA barcode.,Apply the ITS2 barcode to authenticate medicinal materials 皮类药材的条形码鉴定,Phylogenetic tree constructed with the ITS/ITS2 sequences,31 medicinal materials samples,Stability and Accuracy of the Identification using ITS/ITS2 barcodes (Notopterygii )羌活药材ITS/ITS2条形码鉴定及其稳定性与准确性研究,The NJ trees showed that Qianghuo and its adulterants can be easily differentiated according to their monophyly,Xin T Chen S *, et al. Acta Pharm Sin. 2012, 8,Use of the potential DNA barcode ITS2 to identify herbal materials 全草类DNA条形码鉴定,The ITS2 barcode could distinguish successfully all the 34 herbs from their 111 adulterants.,Figure 1. The presence/absence of barcode gaps between herbs and their adulterants.,Pang XH, Chen SL* et al. J Nat Med. 2012,建立中药材DNA条形码鉴定系统网站,http:/www.tcmbarcode.cn,DNA barcode system shows strong abilities in identifying herb cryptic species BLAST PROCESS,打开网址，进入中药材DNA条形码鉴定系统主页,点击物种鉴定，选择植物类药材鉴定（ITS2/ITS）进入右图界面。,在空白处粘贴入需要校对的序列（Query）点击左下角的提交,比对序列相似性信息100.0%为人参，说明该序列鉴定结果为人参,Quick-and-easy DNA identification of herbs is under way,华润三九中药材DNA条形码鉴定专用软件,2019/6/20,七叶皂苷注射剂娑罗子药材的基原植物为七叶树Aesculus chinensis、浙江七叶树Aesculus chinensis var. chekiangensis和天师栗Aesculus wilsonii,娑罗子药材DNA条形码鉴定,柴胡 Chaihu,正品：为伞形科植物柴胡Bupleurum chinense DC.、狭叶柴胡B. scorzonerifolium Willd.的干燥根。 近缘种：同属植物丽江柴胡B. rockii，小叶黑柴胡B. smithii var. parvifolium ，抱茎柴胡B. longicaule var. amplexicaule，细茎有柄柴胡B. petiolulatum var. tenerum 和窄竹叶柴胡B. marginatum var. stenophyllum。,（三）柴胡与其近缘种的NJ树图所示：柴胡正品基原柴胡与狭叶柴胡均独立聚为一支，与其混伪品可以区别开。,结果表明：基于ITS2序列能很好的区分柴胡及其近缘种,（一）柴胡及其及其近缘物种ITS2序列的二级结构：,（二）柴胡与其混伪品ITS2序列间存在较多变异位点,柴胡不同基原ITS2 条形码consensus 序列,柴胡ITS2序列峰图,常用中药材进行了DNA条形码研究,课题组已经进行DNA条形码研究的常用中药材包括：人参，西洋参，三七，大黄，石斛，重楼，金银花，丹参，天南星，红豆蔻，肉苁蓉，京大戟，麻黄，高良姜 ，何首乌，吴茱萸，连翘，甘草，玉竹，冬虫夏草，羌活等,Published papers on DNA barcode,PLoS ONE, 2010,5: e8613,在google scholar中搜索DNA barcode 共2万条 前3篇 有1篇是中国发表。,DNA Barcoding: a hot research topic,中药材DNA条形码分子鉴定体系受到国内外广泛关注,国家药典委员会、国家科技部、国家濒危物种进出口管理办公室等立项研究 德国、英国、美国同行对中国条形码研究给予高度肯定 中国食品药品检定研究院、中国检验检疫科学研究院、国家公安部物证鉴定中心等不同系统均投入人员与资金 深圳华润三九、武汉爱民制药、牡丹江友搏药业、成都新荷花药业、广东王老吉等20余家企业转让应用,举办四次条形码高级研修班 全国18省72家单位200人参加培训,突破传统分子标记内在鉴定能力瓶颈，绕开核基因组的高复杂度，开发种群水平的、物种特异鉴定标记,平台技术的建立具有典型的示范效应，可推广到所有多基源和多产地用药的中药品种,提出“利用叶绿体基因组进行中药材道性鉴别”的学术假设,中药鉴定学与叶绿体基因组学的有机结合,Nature Comms: Genome Sequence of the Model Medicinal Mushroom G. lucidum 自然子刊：基因组解析提出灵芝为首个药用模式真菌,USA Today: Chinese “mushroom of immortality” genome mapped 今日美国评述报道,论文被Nature China选为中国最佳研究推介,Acknowledgements,