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中药鉴定创新方法体系的建立与推广DNAbarcodingsystemforherbalmaterials陈士林ShilinChen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Hoangetal.ScienceTranslationalMedicine,2013,5:197ra102.NortierJL,MartinezMCM,SchmeiserHH,etal.NewEnglandJournalofMedicine,2000,342(23):1686-1692.关木通百度百科木通中药材天地网DNAbarcodingcanachievearapidandaccurateidentificationforherbsApowerfultoolforidentifyingmedicinalplantsandtraditionalChinesemedicinalmaterialsAbruscantoniensisAbrusprecatoriusMedicinalplantsChineseherbalmedicinesDNAbarcodingtechnologyusesashortandstandardizedgeneregiontoidentifyspeciesOnesequenceOnespecies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PLoSONE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。被德国专业网站选为近25年来在ITS2领域最受关注的16篇文章之一该文发表后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Relativedistributionofinter-specificdivergencebetweencongenericspeciesandintra-specificvariationforfourloci.Thetwobarsineachboxrepresentinter-specific(above)andintra-specific(below)geneticdistances.IdentificationofthefamilyRosaceaeW+W-RelativeRanks,n,PvalueResultITS2matKW+=703,W-=0,n=37,P≤1.139×10-7ITS2matKITS2rbcLW+=703,W-=0,n=37,P≤1.137×10-7ITS2rbcLITS2rpoC1W+=703,W-=0,n=37,P≤1.133×10-7ITS2rpoC1matKrbcLW+=620,W-=46,n=36,P≤6.479×10-6matKrbcLmatKrpoC1W+=655,W-=11,n=36,P≤4.174×10-7matKrpoC1rbcLrpoC1W+=359,W-=106,n=30,P≤0.009rbcLrpoC1Wilcoxonsigned-ranktestsofinter-specificdivergenceamongloci.ITS2andrbcL,matK,rpoC1werecompared.•For54samplesfrom43speciesin19diversegenera,thesuccessfulidentificationratewithITS2was100%,followedbymatK(94%)andrbcL(65%).•For1410samplesfrom893speciesin96generaNo.ofgenera(≥2species):47(1351samples)No.ofspecies(≥2samples):297(814samples)IdentificationratewithITS2was100%and78%atgenusandspecieslevel,respectively.XPang,ShilinChen*.Cladistics,2011,27ComparativeanalysisofalargedatasetindicatesthatITSshouldbeincorporatedintothecorebarcodeforseedplantsChinaPlantBOLGroup,LiDezhuetal.,PNAS,2011WethereforeproposethatITS/ITS2shouldbeincorporatedintothecorebarcodeforseedplants.(建议ITS/ITS2应成为种子植物的核心条形码)IncaseswhereitisdifficulttoamplifyanddirectlysequenceITSinitsentirety,justusingITS2isausefulback-upasitiseasiertoamplifyandsequencethissubsetofthemarker.(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、PLoSONE、PNAS、BMCEvolBiol、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混伪品及近缘种人造伪品：滇紫菀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：Cordycepsrobertsii（新西兰）结果：基于BLAST/DISTANCE的鉴定效率：100%基于NJtree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XiangL,ChenSL,etal.FEMSmicrobiologyletters,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Morphologicalcharacteristicsofthepowder,decoctionpieces,seed,seedling,andfibreamongtheP.ginseng,P.quinquefolius,Pnotoginsenganditscloselyrelatedspeciesaresimilar.Itisdifficulttoidentifysomanydifferentpanaxspeciesandginsengproduceinmedicinalusesbytraditionalclassificationmethods.UsingDNAbarcodetoidentifypanaxmedicinalspecies人参类及其混伪品的鉴定1315101520253035404550SN01:p.ginseng;SN03:p.quinquefolius显示人参、西洋参ITS2序列的SNPs位点信息。Position32and43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份应用ITS2序列两个SNPs可以快速准确鉴别人参西洋参，所得组合为C-T，则确定为人参，所得组合为T-C，则确定为西洋参ITS2sequenceposition1-50ofp.ginsengandp.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.AFastSNPIdentificationofMedicinalPanaxSpeciesBasedonDNABarcodingAnalysis,14金银花药材及其混伪品的ITS2条形码鉴定金银花（忍冬）山银花（红腺忍冬）山银花（黄褐毛忍冬）山银花（灰毡毛忍冬）研究对象：包括24份金银花药材。M:marker1、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25mg，利用植物基因组DNA提取试剂盒(TiangenBiotechCo.,China)提取总DNA。ITS2序列PCR扩增程序及电泳图94℃变性5min94℃变性30sec58℃退火30sec72℃延伸45sec72℃延伸10min40个循环BLAST鉴定IdentificationofCortexHerbsSunZY,ChenSL*.JNatMed.2012.Theidentificationefficiencyof51samplesbelongingto19kindsofcortexherbsinChinesePharmacopoeiaexaminedwas100%usingITS2sequenceasDNAbarcode.ApplytheITS2barcodetoauthenticatemedicinalmaterials皮类药材的条形码鉴定条羌N.franchetii大头羌N.franchetii蚕羌N.incisumPhylogenetictreeconstructedwiththeITS/ITS2sequencesadulterantsITSITS231medicinalmaterialssamplesStabilityandAccuracyoftheIdentificationusingITS/ITS2barcodes(Notopterygii)羌活药材ITS/ITS2条形码鉴定及其稳定性与准确性研究TheNJtreesshowedthatQianghuoanditsadulterantscanbeeasilydifferentiatedaccordingtotheirmonophyly..ITSITS2HaplotypeVariablesitesHaplotypeVariablesitesN.incisum4433N.franchetii61045XinTChenS*,etal.ActaPharmSin.2012,8UseofthepotentialDNAbarcodeITS2toidentifyherbalmaterials全草类DNA条形码鉴定TheITS2barcodecoulddistinguishsuccessfullyallthe34h