陈士林——中药DNA条形码鉴定方法体系的建立与推广

中药DNA条形码鉴定方法体系的建立与推广陈士林中国中医科学院中药研究所中药鉴定领域寻求变革与突破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--关木通误当木通广防己误当防己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DNAbarcodingcanachievearapidandaccurateidentificationforherbsApowerfultoolforidentifyingmedicinalplantsandtraditionalChinesemedicinalmaterialsAbruscantoniensisAbrusprecatoriusMedicinalplantsChineseherbalmedicinesDNAbarcodingtechnologyusesashortandstandardizedgeneregiontoidentifyspeciesOnesequenceOnespecies•PlantWorkingGrouprecommendedrbcL+matKastheplantbarcode,successfullydiscriminateatthespecieslevel--72%.•Farfromperfectduetothelowidentificationrate•PNAS2009Science2009ThreegenomesinplantsandcandidatebarcodesfocusedongenesandintronsofthechloroplastgenomeIdentificationefficiencyforITS2:92.7%and99.8%atspeciesandgenuslevel,respectivelyMarkerMethodofspeciesidentification(Rossetal.2008SystBiol57:216-230)CorrectidentificationIncorrectidentificationAmbiguousidentificationSpecieslevel%Genuslevel%Specieslevel%Genuslevel%Specieslevel%Genuslevel%ITS2BLAST92.799.8007.30.2Distance90.399.7009.70.3psbA-trnHBLAST67.695.40032.44.6Distance72.896.50027.23.5ForITS2,basedonanalysisof6600plantsamplesbelongingto4800speciesfrom753diversegeneraof193familiesin7phylaForpsbA-trnHspacer,basedonanalysisof2000plantsamplesbelongingto1433speciesfrom551diversegeneraof135familiesin4phylaTheITS2locusisafavorableuniversalbarcodeandpossessesaperfectprospectinauthenticatingspeciesusedforawidesetofplanttaxa.ThepsbA-trnHspacermaybeacomplementarybarcodetobeused.ShilinChen*etalPLoSONE2010,5(1):e8613IdentificationefficiencyforITS2andpsbA-trnHlociusingdifferentmethods课题组首次提出并验证ITS2条形码，引起国际广泛关注国际期刊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319次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)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XiangLi,ChenShilinetal.FEMSMicrobiologyLetters,2013DNA条形码鉴定冬虫夏草及其伪品IdentificationofLyciumbarbarumanditscloselyrelatedspeciesusingITS2barcodeFoodResInt.2013,Fig.1ComparisonofLyciumbarbarumandL.chinensecollectedfromdifferentprovinceinChina57medicinalmaterialssamplesFig.2TheNJtreeofLyciumbarbarumanditscloselyrelatedspeciesconstructedwiththeITS2sequences.Thebootstrapscores(1000replicates)areshown(≥50%)foreachbranch.Theinter/intra-specificdivergence(Table1),NJ-tree(Fig.2),andthevariablesites(Fig.3)demonstratedthattheITS2barcodecandistinguishLyciiFructusanditsadulterantsaccuratelyandswiftly.ITS2DNAbarcoderepresentsanewtechniquefordifferentiatingLyciiFructusfromitsadulterants,thusguaranteeingclinicaldrugsafetyandensuringthevitalinterestsofpatients.Fig.3VariablesitesinthehaplotypesofthethreeLyciumspeciesTable1Analysisofinter/intra-specificdivergenceinITS2barcodeofthethreeLyciumspeciesmean±SD(range)Averageinterspecificdistance0.0535±0.0114(0.0269-0.0892)Averageintraspecificdistance0.0043±0.0064(0.0000-0.0844)Intraspecificdistanceso