DB45_T 1856-2018 蛤蚧及其伪品COI条形码鉴别技术规程(广西壮族自治区).pdf

ICS 65.020.30 B 38 广西壮族自 J曰DB45 区地方标准DB 45/T 1856-2018 蛤蛤及其伪晶COI条形码鉴别技术规程Identification of Gekko gecko Linnaeus and Adulterants Using the COI Barcode 2018-10-20发布2018-11-20实施广西壮族自治区质量技术监督局发布DB45/T 1856-2018 目次前言.11 l 范围.1 2 术语和定义.1 3 较别程序.2 附录A(规范性附录)蛤盼伪品.3 附录B(规范性附录)蛤蛤及其伪品COIDNJ序列.4 I 目IJ1=1 本标准按照GB/T1.1-2009给出的规则起草。本标准由广西壮族自治区卫生和川划生育委员会提出。本标准由广西卫生标准化技术委员会归口。本标准起草单位:广西壮族自治区药用植物园。DB45/T 1856-2018 本标准主要起草人:徐永莉、李力、问志刚、张月云、赵成坚、黄勇、谷颖乐。II DB45/T 1856-2018 蛤阶及其伪品COI条形码鉴别技术规程1 范围本标准制定了蛤蛤(白，kkogeko)及其伪品的COI条时马鉴别m语和定义、鉴别程序。本标准适周于广西境内蛤蛤及其伪品COI条形码的鉴别。2 术语和定义r列术语和定义适用于本文件。2.1 蛤町Gekkogecko 蛤蛤(Gekkog，cko)属爬行纲Reptiha、有鳞日Sq&arr正a臼叫amat蛤药材为除去内脏的干燥体。包括时时前口红点蛤蜕2.2 去蛤町I伪品Gekko gecko adultera时S市场上当蛤蛤销售和使用的动物。详见附录Lj 2.3 lIl,111111 11安慰DNA条形码DNA barcoding 嚣ml四生物体内能够代表该物种的标准的F有足够变异的、易扩增且较短的DNA片段。2.4 Jqj COI 细胞色素c氧化酶亚基1(cytochrome c oxidase subunit ICOI)，线粒体基因组的蛋白质编码基因。2.5 COI条形码COI barcoding 集中存在于动物线粒体细胞色素C氧化酶亚单位1(coI)特定区段的一段大约有650个碱基的DNA序列。2.6 近邻相接法neighbor-joining method(NJ法)一种研究DNA而建立亲缘关系的方法，由序列建立支序图或亲缘关系图。D845/T 1856-2018 2.7 l陆基对。3 鉴别程序3.1 鉴别条件PCR扩增仪、核暇电泳仪和水平电泳槽、)tM交成像系统、纠f只基因提取J剂市、序列比对软刊。3.2 引物序列LCOI490:5-GGTCAACTCATGATATTGG-3。HC02198:5一TCTTCAGGGTGACCATCA-3。3.3 DNA的提取取药材样品，75%乙醇表面消毒，用灭商手术刀，取样品皮下肌肉约30吨，剪町，DNA提取研磨仪研磨2mi日，用组织基因提取试剂盒提取总DNA，提取步骤参见各产品使用说明节。3.4 PCR 扩增反应体和(为251，所加试剂包括5U/L Taq DNA聚合酶CTaKaRa)O.51，10 mmol/L dNTP 11，10 pmol/L上下游引物各O.51，10 X PCR buffer 2.51，Mg2 1.51，DNA模板1LoPCR反应在PCR扩增仪上进行，反应参数为95C3min,C94C 45s,55C 45s,72C 45s)X39,72C 5min,4C 保存。3.5 电泳将PCR产物5L与上扬染料3L混合，加入1.5%琼脂糖凝肢的点样孔中，同时设100bp梯度DNA标准分子量作为片段大小的标准。100V电压电泳约40mino 3.6 成像及分析将电泳后的琼脂糖凝胶置于凝胶成像系统观察窗内，观察分析并成像保存。3.7 DNA样品测序j疑胶上出现一条600bp大小的特异性条带PCR扩增产物用试剂盒对PCR扩增的样品进行纯化，纯化的步骤参见各产品使用说明书，将检测纯化后的样品进行测序。3.8 鉴到将所得序列经计算机拼接辅以人工校对，采用Biast序列相似性搜索比对，蛤自C0I5端序列，即蛤蛤DNA条形码序列，562 bp的长度用于种问水平的分析。蛤蛤同其伪品间的序列差异为6.0%64.0%,平均序列差异为35.0%。通过NJ法并利用实验序列和GenBank序列所构建系统聚类树。基于COI序列构建的NJ树将蛤蛤与伪品区分。2 附录A(规范性附录)蛤蛤伪品DB45/T 1856-2018 无联壁虎Ukkoswinhoni s、中国凛蜘Paral11esotritonchinensis、多庇壁虎Gekkojaponicus、1金虎Goniurosauruslichtnfeld，r1、西藏沙蝴iPhrynochalus拍baldi、红蝶庇版TylototritonVrrucosus、贵州|疵蛛Tylototriton付出:chowensis、岳山岩蝴iLaudakiahimalayana、变色树虫f)jCalotS队.、JVrsicolor、青海沙蚓jPhrynoc叩h叫usv7angal-ii、山虞酣Batrachupruspinchonii、东方蛛幌Cynopsorientalis等。3 DB45/T 1856-2018 附录B(规范性附录)蛤蛤及其伪晶COIDNA序列B.1 蛤蛤Gekkoch nens s 562 bp gaattaagtc aaccaggagc actccttgga aacgaccaac tatacaatgt aatcgtaaca gcacacgcat ttgtaataat cttctttata gtgatgcctg ttataatcgg ggggtttggc aattgattag tccccctaat aattggcgca ccggacatag cttttccacg aataaacaac ataagcttct gactgctccc cccagcatta ttcctcctac tagcctccgc gagtgtgga agctggggcgg ggacaggatg aactgtatac cccccactag cagctaatct agcacatgca ggcgcatctg tagacctggc catcttttcc tt正acacctagctggagtctc ttcc正atcttaggggccatca attttatc正acc正acctgcattaatataa，a ccccaagcat aacccaatac acgacacccc tgtttgtatg atcggttcta attactgccg tactacttct cctagcacta ccagtgcttg ccgccggcat cactatactg ctgaccgacc gtaaccttaa cacatcattc tttgaccctg ccggaggcgg gg B.2 无躁璧j竞Gekkosw nhon s 825 bp atgtttaccc gttgatttct ctcaacaaa agtgggcaca gcactgagct tgttaattcg cgcggagtta agccagcctg gggcactact aggggatgat cagatctata atgttatcgt aacagcccat gcattcgtaa taattttttt tatagtcatg cccgttataa ttggcggctt cggcaattga cttgtaccac ttataattgg cgcacccgat atggcttttc cacgaatgaa taatataagt ttttgactgc tcccaccatc actgctacta cttctcgctt cttccggagt tgaagcaggt gctggaaccg gttgaactgt ctatccccca ttagcagcta atcttgcaca tgcgggcgca tcagttgacc tagttatttt ctctctacat ttggcaggtg tatcttcaat cttaggggct atcaatttta tcaccacctg cattaatata aaatctactg ccatgtcgca gtacaatacg cccctattta tttgatcggt attaattacc gccgtactac tcttacttgc cctaccggtt ctggctgccg gaattactat acttctaacc gaccgcaacc ttaacactac attctttgac cctgcggggg gcggagatcc tgtactatat caacacctat tctgattttt tgggcaccca gaagtatata ttttaattct tcccggattc ggcatagtat cgcatatcat tacgtactat gcaggaaaaa aagaaccgtt cggctacatg gggatagtct gggct B.3 中国攘蠕Paramesotr ton ch nens s 2690 bp gttaaggtgg cagagcccgg taattgcaaa agacctaagc cctttccacc agaggttcaa ttcctcttct taactgtgta tactatatcc gccctcttaa acccattact atatattatc ccagtattat tagccgtggc ctttttaact cttgttgaac gaaaagtact agggtatata caactgcgga aaggaccaaa catagtgggg ccagtaggcc ttcttcaacc aatcgcagat ggactaaaac tatttattaa agaacccgta cgcccatcaa catcgtcaca aactct gttt ctcattatac ccataatagc gctaactctg gcactaatta tttgaatccc actacccatg ccctttacac tgtctaatct aaatct gggg gttcttttcc ttctatccct atctagcctg gccgtatact caatcctagg gtccggctgg tcgtcaaact caaaatatgc cctagttgga gcacttcgcg cagtagcaca gactatctca tatgaagtta cactaggact aatcctacta tgccttattt tactaacagg gggcttcata ttaacaaact ttaatatctc gcaagaatca acctgattta ttattccagc atgacctata gcaacaatgt gatttatctc acccctagca gaaacaaacc gaaccccatt tgatctaaca gagggggaat cagaactagt ctccggattt aacgtagaat atgcaggagg gccattcgca ctattttttc tggcagaata ttccaacatt ttacttataa ataccctatc agctatccta ttccttggcc aaacaacaag ccacattcaa ccagaaatat caacaattaa cctaattatt aaagcatcga 4 DB45/T 1856-2018 cacLaLcaaL cgLaLLLcLc LgaaLLcgag caLccLaccc acgaLLLcgg LaLgaLcaac LaaLacacLL aaLcLgaaaa aacLLcLLac cccLaacaaL LacaaLaacL cLcaLacaca LcLcacLacc aaLLacaaLa gcLggLaLcc ccccLLLaLc cLaggacgLg Lgcccgaaag aLaggggcca cLLLgaLaga gLggccgaLa gaggLLcaaa cccLcLcaca LcccacLaaa aaaacaggac LcgaaccLaL cccLagagga Lcaaaacccc LcgLgcaccc acLacacLaL cLLLLaaggL aaaaLaagcL aaaLcaagcL LLLgggccca Laccccaaac aLgLLggcga aaacccLLcL LLLacLaaLg agccccLaLa cacLgLccaL aLLaaLaLca agLcLLgccg LaggaacaaL LaccacacLa LcaagcLaLc acLggLLLcL agcaLgaaLa ggacLagaga LcaacacaLL ggcLaLLaLL ccacLgaLaa cLaaaaLgca ccacccccgg gccacagaaL cLgcaacaaa gLacLLLcLa acacaagcca tagcctccgc cttaatttta ttctcaEleaa ttattltitlegcatgc飞attgtgaca ggagaELtgtiti CElateEltaag cataaatcac catatatcaa caaca咀tLftcatiil t EtactIattgct ttElgcultta aagtItIt cgcaecattt ctleetEltgat ucmgacgt tcucaaggcAadi7EC川L二1币aUGtJ cE t11gccLJ飞atcctgtca acatg刷刷刷ttagcccccatagcctta ctCElttttga caa gccacactaaElCOg仁atad a a ctaa tattititagcactElactj号tccatgcttgtag ggggatgagg gggcctaaat caaacgcaa cacgaaa ccttttcccc caatctagaca g ctactaaacc飞attagtatacaaBta tatLctg acctcatetda a t tgtttataac actaataacc ttaaattcaa caaacat a caaactctca Caa ectcgtgac t aaacacc aacactati g a gcctttacaa taattgttct aatggccctt ggagggctcc cacft cecacat ggattttta ccaaaatgac taattt Ca agaaataacc aaacaacacc tcaatgcact ctccgccaca atagccatat tagccbtact cagcctattt ttttatcta飞gtttatcatatacaatctca ataacctcac cc川叮tat ctcaaactca ageetaaatc tt gacggaaaaa aacaMeeta川ataatcccaattataat tatattgtca gcaatat aa taceaataac t,ccaac tg ctaataataa actaaggactaztcac ggataaca agaccaaaga ccttcaaagc cttaagcaga agttaaaatc ttctagtcct tgacaaaacc tp gcaggatac t ccacat cttctgaatg caacccagac actttaatta agctaaagcc ttctagatta gca挝ctttgaccctacaalc t cttttagtta acagetaaat actcaatcaa cgagctttaa tctacttctc og、在tgttgagaaaaaaag?gggagaaF cccggcaaaa gctactttgc tCHUmat ttgCMUtt atatgeum cateacagt ag cttggtaaaa aaaggact:1币aacctttatgacaagggcta macettgca cctgcttcgg ccattttace aW gtr gataa C actcgatgac tlattttctaJ飞B.4 多疵璧虎Gekkojaponcus 825 bp，1/11 可1I同atattcaccc gttgatttct ctcaacaaac cataaggata tcggcacact ctatttat g tttggtgtct gggccggcat 部同gcaccgcattaagtc山:tat问cg.叫mtgaa itcagmg向CMrc F ct c如gacgatcagatttaca acgteattgt caccgcecac gcattcgt aa taattttttt t tagtaata ccggteatgaC i ftcggcggctt cggtaattga ctcgtacccc ttataatcgg cgcacct a gf a g c atggccttcc cacgaataaa caaca也agttctgacttc tgcccccctc tctactactt ctactcgcct cctctggagt ggaggccggc gctggcaccg gttgga,tgt atacccccca ctcgcagcaa acctcgcaca cgccggcgCCgA ttccgtcgacc tcgttatct t t teattgcat ttggccgL ggg tctcgtcaat tcttggtgca atcaacttca ttaccacctg tattaatafa aaatccacac ctataacaca ata巳aaacgcccctgttta tttgatcagt tctaatcact gccgtactac tcttgctcgc tttgccagta ttagcg主ccggaat/taccat acttctaacc gatcgtaatc tcaacaccac tttttttgat ccagctgggg gcggcgaccc cgtactgtat caacatctat tttgattctt cggtcaccca gaggtatata ttttaattct ccccggtttc ggcatagtat ctcatattat cacatattat gcagggaaaa aggagccctt cggctacata ggaatggtct gagcc B.5 险j竟Gon urosaurus I chtenfe I der 562 bp gagcttaacc agcctggggc gctcctcggc gacgaccaaa tttacaatgt aatcgtaacc gcccatgcct ttgtcataat ttttttcata gtaatacctg ttatgattgg gggcttcgga aactggctag tacccttaat aattggggcc cctgacatag ctttcccccg aataaacaat ataagcttct gacttttacc cccatcttta cttctactcc tagcttcttc aggtgtagaa 5 DB45/T 1856-2018 gclgglgclg gaacaggglg gaccglalac cccccaclag ccgcaaaccl cgcacacgca ggagcalccg lagaccllac aalcllllcc clacalclcg ccgglglllc clcaallcla ggggcaalla acllcallac lacclglall aacalaaaac cccclgcaal alcacaalal aacaccccgl 19l1lglglg alcagllcla alcacagcag laclaclacl lclalcacla cclglacllg ccgclggcal lacaalacla claacagalc gaaacclaaa cacggcllll lllgalcclg cagggggagg gg B.6 西藏沙晰PhrynocPhalusthobaldi 661 bp acactttact tcctattcgg caccatagcc ggcttcactg gctcaacagt cagccttatg attcgactac aactaattca acccggccaa cacaacggag acacaatata caacactttc attacacttc atgccttcat tataatcttc tttatagtaa taccaatcat aatcggagga ttcggcaact gattagtacc actcatacta ggcgccccag acatagcatt tccacgaata aataacataa gcttctggct cctaccacca tcgttctttt tacttctaat gtcatcaaaa ttcggcgacg gagtaggaac aggatgaaca atttatcctc cattagcagg aaatattgca cactcaagcc catcaataga cataacaatt ttcgcactac atctagccgg agcatcctca atcttggggg ccattaactt tattacaacc tgcattaata tagccccaca ctcaacaacc ccatacaact gaccactgtt cgtctgatcc gtattcctta ccgcaatctt actcctactg gccatcccag tattagcagc agccatcacc atgcttctaa cagaccgaaa cctaaacaca acattcttcg accccgccgg aggaggagac ccaattctct tccaacacct a B.7 红攘疵邮票乃lototriton阳rrucosus2889 bp cgctattaag gttcgtttgt tcaacgatta aagtcctacg tgatctgagt tcagaccgga gtaatccagg tcagtttcta tctataaaaa attttttcta gtacgaaagg accgaaaaaa tagagcccat tactaaacaa tgctcttaca cagttgaaaa caaataaaac acgcacacaa aacacacccc caagaacagg gtaaagttaa gatggcagag cccggtaatt gcaaaagacc taagcccttt cacccagagg ttcaactcct cttcttaact atgcatatcc tacaaacact catcaaccca ttactatata ttattcccat cttattagct gtagcattcc tcacactagt cgaacgaaaa gtattaggat atatacagct ccgcaaaggc ccaaacatcg taggaccaat aggactatta caaccaatcg ccgacggagt aaaactattt attaaggaac ccatccgacc atccacatcc tcacagacct tatttatcac aatgcctata atagcactaa cactagcact aatgatctga attccccttc caatgccatt tatactatca agccttaacc taggagtact gttcatatta gccatatcaa gcttagctgt atactcaatc ttaggctcag gatgatcatc caactcaaaa tatgcactaa tcggggcgct acgcgcagta gcacagacca tctcatatga agtaacacta ggacttattc tcctctgtct tattttaatg acaggaagct ttacattaat aagcttcaac acactacaag aaccaacatg acttatcata ccagcatgac caatagcaac aatatgattt atctcaacat tagcagaaac aaatcgagcc ccatttgatt taacggaagg agaatcagaa ctagtatcag gctttaatgt agaatacgcc ggaggaccat tcgcactatt ttttctagca gaatactcaa atatcttact aataaatgcc ctctcaacta tcttatttct aggaacaaca attaatcacc tacaaccaga aatacttaca attaacctaa taataaaagc atccgcccta t caattatat ttctatgagt acgagcatca tatccacgat tccgctatga ccaattaata caccttatct gaaaaaactt cctcccaat t acaattggac ttacactaat acatatctca ctaccaattt taacctccgg aattccctct gcactatagg acatgtgccc gaaagatagg gcttactttg atagagtaac acatagaggt tcaaaccctc tcatctcctg tataaaaaaa taggatttga acctacccca aggagatcaa aacccctcgt gcttccacta caccactttt tactagtaaa ataagctaaa ctaagctttt gggcccatac cccaaacatg ttggttaaaa tccctctttt actaatgaat ccatatgcac tatcaatcct gttgtcgagc ctagcagtag gaacactcac aactctctca agctcccatt gattcctggc atgagtagga ctagaaatta acacactagc aataaccccc ttgataacaa aaatacacca cccacgatca acagaatcag ccacaaaata cttcctaacg caagccaccg catcagcctt aatcttattt tctacaatca caaacgcatg agccactgga gaatgaacaa ttaccaccat aaatgacaac acatcaacat ttatactaac aattgcccta gctattaaac ttggaatcgc cccttttcat ctatgactac cagacgttat acaaggacta aatctaacaa 6 DB45/T 1856-2018 calgcclaal ccllgcgaca 199caaaaal lagccccaal glccllaala allalaacca accaccaall aaacacaaac algclaalla caalagccgl gllalcaaca glaallggcg galgaggggg ccllaaccaa acacaaallc gaaaaalaal agcalaclca lclalcgccc acclcggalg aalaacacla glcclalcll lclccccaaa lllaacaacc cllaacclaa laalclaccl aalaclaaca lcalcaalal lcllaalaal aaaaacccla aaclcaacaa alallaacaa aclllcaala lCllgaclga aagccccccl allagcalca lcaalaalaa llacaclcal agccclagga gglclacccc caacalcagg gllcclacca aaalgaclla lcclacaaga aalaaccaaa caacacclag gcaccglalc cacaclaala gclalalclg cgclaclcag lllallcllc lacclacgac lalcalalgc aalclcacla accaccacac caaacacclc aaaclccaac ataac叫acgaata accaacacta ctagcaa tleettcta gt ctttgEltaag cceca FE ctattettctgaatgcv c d fa e-atc 7 c i eca gtI Ca cttttiti tttitigctaa g accttctaga ctagcaggct ctaacccgc ga.cttttag ttaacagcta aacgGtcaat ccaacaagct ttagtctact gaacatc acaagg叫ggtaaaaaaag,gggtttaacc t山阳gggcaa毛时cacctact问gccacttaccagtg 远在生iB.8 贵州疵师、Ty/ototritonkWI町chowensis 566 bp g阴阳叫gca叫cactaagcctac同tI时出agcgaact叩:caaccaggtgct叫阳cg叫t叩tctataat归叫tta叫ctcatgcct叩M副ttt叮Ct叫gtaataccagt削剧tcgggggattcggaa actgactctt accgctaata attggggccc C，;正在agatatggc cttcccccga ataaataaca taagcttctg actcctcccg ccatcattcc tgctcct时tgcctcgtcaagaag ctgi t ggcggg、accgggt严actgtataccccccattagc tggtaatctt gctcacgcag gagcatcagt tgacctcacc attttctccc tccacctcgc aggcgtatct tcaatccttg gtgcaattaa ctttattacc acatcaatta ac仇c tztaaaacc cecaf teat i lamE tcacaatafhv aaacgcccct ttttgtatga tcagttttaa tcacggG川tctgctactcatccctcc ctgtac LghggctggtaTC号ccatgcttttaaccgaccg taatctaaac acaaca I 1I111丁11 lm/矿B.9 喜山岩晰Laudakiahma/ayana 549 bp caattaatcc aacccgga气agtacagtggt.gacictatgt ao倒tacctttattacactc catgccttca tcataatctt ctttatagtc atacccatca taatcggggg attt也gaaactgactagtac cattJ at bgFt aggcgcaeca gacatggcat tcccacgaat aaacaacatg agcttttgao乒tat飞gcccccatcattcttt atactataa tgtcttctaa attcggatcc ggcgttggaa cagggtgaac tatttacccg ccattggcgg gaaacctgac cC51ctcc已ztgc ccateaatag acataaccat ctttgccctt cacctagccg gagcctoa主Aaat，cdcEgggc gcaattaact tc,atgy cca a ac atgcattaac ataagcecac attcaataac cccttacaac atagtaaccctatm巳ztttag、tE a ttgtttgate tgtattcttt act,atct tattactgct cgccatecca gtattagcag cagccatcac aatactttLg accgaccgaa accttaacac gtcattct t t gacccagccg gaggcggag B.10 变色树晰Ca/otesversic%r 658 bp aactttatat ttcttatttg gtgctgcggc aggccttaca ggctcactcg tgagccttct tgtacgggca cagttaatac aaccaggaca agccctaggg ggggattcac tctacaatgt tttcattaca tttcatgcac tggtaatgat tttctttatg gttatgccga ttataatcgg cggcttcgga aactgattag tacctttaat gctgggagca ccagacatag catttcctcg aataaacaac ataagctttt gattactccc cccttcattt cttcttctac tgttctcatc aggctttgaa acgggggtcg gaacggggtg aactatctac cctccccttt ccaacaacat cgctcactcc gggccctcaa tagaccttgc tatcttctct 7 DB45/T 1856-2018 cLacaccLag cLggagcgLc aLcaaLLcLL ggcgccaLLa aLLLcaLcac LacgLgcaLL aacaLaagLc ccagccgcac LLccccaLaL aaLLgacccc LaLLLgLLLg aLccgLLLLc LLLacLgcaa cccLacLLcL gcLLLcccLL ccagLaLLag ccgcLgcaaL cacgaLacLa cLcacagaLc gcaaccLcaa cacaLcaLLL LLLgagccaL cLggaggggg agacccgaLc LLgLLccaac accLaLLc B.11 青海沙晰PhrynocPha/usv/anga/661 bp acactttatt tcctgttcgg cgccatagcc ggcttcactg gctcaacagt tagcctcata attcgactac aactaattca accgggccaa tacaacggag acacaatata taa taccaattat aatcggagga ttcggtaact gactagtacc gctgatacta ggggccccag acatagcatt tccacgaa aataatataa gcttctgact cctaccacca tcgttctttt tacttctaat ctcgtcaaaa ttcggcgacg gagtaggaac aggctgaaca atttatcccc cactagcagg aaacatagca cactcaagcc catcaataga cataacaatt ttcgcactac acctagctgg agcatcctca atcttagggg ccattaactt tattacaacc tgcattaata tagctccaca ctcaacaacc ccctacaact gaccactatt tgtttgatcc gtattcttta ccgcaatctt actcctactg gccattccag tattagcagc agccattacc atgcttctaa cagaccgaaa cctaaacaca acatttttcg accccgccgg agggggagac ccaattcttt tccaacacct a B.12 山溪鱼jtBatrachuprusp nchon 662 bp actctatatt tagtatttgg tgcttgggcc ggaatggtgg gcactgccct a