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Article(id=1241779359137206410, tenantId=1146029695717560320, journalId=1205117023404326918, issueId=1241779355555266850, articleNumber=null, orderNo=null, doi=10.16155/j.0254-1793.2024.05.02, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=null, receivedDate=1681228800000, receivedDateStr=2023-04-12, revisedDate=null, revisedDateStr=null, acceptedDate=null, acceptedDateStr=null, onlineDate=1773992870363, onlineDateStr=2026-03-20, pubDate=1717084800000, pubDateStr=2024-05-31, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1773992870363, onlineIssueDateStr=2026-03-20, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1773992870362, creator=13701087609, updateTime=1773992870362, updator=13701087609, issue=Issue{id=1241779355555266850, tenantId=1146029695717560320, journalId=1205117023404326918, year='2024', volume='44', issue='5', pageStart='737', pageEnd='920', issueExtLink='null', onlineDate='null', pubDate='null', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=null, createTime=1773992869509, creator=13701087609, updateTime=1773992925624, updator=13701087609, preIssue=null, nextIssue=null, ext={EN=IssueExt(id=1241779590985749489, tenantId=1146029695717560320, journalId=1205117023404326918, issueId=1241779355555266850, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1241779590985749490, tenantId=1146029695717560320, journalId=1205117023404326918, issueId=1241779355555266850, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null}, startPage=750, endPage=755, ext={EN=ArticleExt(id=1241779362865942679, articleId=1241779359137206410, tenantId=1146029695717560320, journalId=1205117023404326918, language=EN, title=The ITS2 sequences’ characters study of imported Arnebiae Radix based on DNA barcoding and PCR-RFLP technologies*, columnId=1241779356482208035, journalTitle=Chinese Journal of Pharmaceutical Analysis, columnName=Column on Quality Evaluation of Arnebiae Radix, runingTitle=null, highlight=null, articleAbstract=
Objective:

To provide reference for quality control and authenticity identification of Arnebiae Radix medicinal materials and decoction pieces in the market. By studied on the ITS2 sequences’ characters of imported Arnebiae Radix，based on DNA barcoding and PCR-RFLP technologies.

Methods:

The ITS2 region was selected as the DNA barcode sequence for comparison and identification of imported Arnebiae Radix and reference medicinal materials. The ITS2 sequences of imported Arnebiae Radix from different sources with reference medicinal materials were compared based on DNA barcoding and PCR-RFLP technologies.

Results:

After the restriction endonucliase AluI enzyme digestion，the agarose-gel electrophoresis results of 39 imported Arnebiae Radix samples showed that，only DH3 had bands at around 500 bp，and none bands between 100 bp and 300 bp. And the results of other imported Arnebiae Radix samples had two or three obvious bands between 100 bp and 300 bp. The ITS2 sequences of imported Arnebiae Radix samples were compared with the reference medicinal materials，among which DH3 had the largest differences of 15 bases compared to the reference medicinal materials，the ITS2 sequence of F2 was same to the reference medicinal materials，and other imported Arnebiae Radix samples had 1-9 bases difference compared to the reference medicinal materials. The clustering results showed that the imported Arnebiae Radix sample DH3 was clearly distinguished from other imported Arnebiae Radix samples and reference medicinal materials which was in a single branch. There were 14 samples，which were clustered together with the reference medicinal materials in one branch with support rate ≥50%.

Conclusion:

The ITS2 region is selected to compare the similarities and differences of ITS2 sequences between imported Arnebiae Radix samples and reference medicinal materials based on DNA barcode and PCR-RFLP technologies，which provids a reference for effective identification of Arnebiae Radix medicinal materials and decoction pieces，and a strong guarantee for market supervision of Arnebiae Radix medicinal materials.

, correspAuthors=Jian ZHENG, Shuang-cheng MA, authorNote=null, correspAuthorsNote=null, copyrightStatement=null, copyrightOwner=null, extLink=null, articleAbsUrl=null, sourceXml=null, magXml=null, pdfUrl=null, pdf=null, pdfFileSize=null, pdfExtLink=null, richHtmlUrl=null, mobilePdfUrl=null, reviewReport=null, pdfFirstPage=null, abstractGraph=null, abstractGraphContent=null, abstractVideo=null, citation=null, cebUrl=null, magXmlContent=null, mapNumber=null, authorCompany=null, fund=null, authors=null, authorsList=Jie LIU, Sheng-yun DAI, Hai-yuan GU, Fei QIAO, Chao-jie LIAN, Li-nong GUO, Jian ZHENG, Shuang-cheng MA, Jia MI), CN=ArticleExt(id=1241779363671249083, articleId=1241779359137206410, tenantId=1146029695717560320, journalId=1205117023404326918, language=CN, title=基于DNA条形码和PCR-RFLP技术的进口紫草ITS2序列特征研究*, columnId=1241779358067654949, journalTitle=药物分析杂志, columnName=紫草类民族药材的品质评价研究专栏, runingTitle=null, highlight=null, articleAbstract=
目的：

基于DNA条形码和PCR-RFLP技术研究进口紫草ITS2序列的特征，为市场紫草药材和饮片的质量控制与真伪鉴别提供参考依据。

方法：

选用ITS2区域作为对进口紫草和紫草对照药材进行比较、鉴定的DNA条形码序列，并基于DNA条形码和PCR-RFLP技术比较不同来源进口紫草的ITS2序列与紫草对照药材的异同。

结果：

39份进口紫草样品经限制性内切酶AluI酶切后，其产物的琼脂糖凝胶电泳检测结果显示，仅DH3在500 bp左右有条带，而在100~300 bp无条带，其余样品均在100~300 bp有2条或3条明显条带；进口紫草样品与紫草对照药材的ITS2序列进行比对，其中样品DH3与紫草对照药材的碱基差异最多，有15个碱基差异，样品F2与紫草对照药材的ITS2序列一致，其他进口紫草样品与紫草对照药材的碱基差异为1~9个碱基；从聚类结果中可以看出进口紫草样品DH3与其他进口紫草样品和紫草对照药材均明显区分，独自为一枝，而与紫草对照药材共同聚为一枝且支持率≥50%的样品共有14个。

结论：

选用ITS2区域，基于DNA条形码和PCR-RFLP技术，比较了进口紫草与紫草对照药材ITS2序列的异同，为紫草药材及饮片的有效鉴定提供参考依据，为紫草药材的市场监管提供有力保障。

, correspAuthors=郑健, 马双成, authorNote=null, correspAuthorsNote=
** 郑健　Tel：（010）53852080；E-mail：;
马双成　Tel：（010）53832076；E-mail：
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Tel：（010）53851401；E-mail：

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Tel：（010）53851401；E-mail：

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Tel：（010）53851401；E-mail：

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(Boraginaceae) and related taxa：evolutionary relationships and character evolution, refAbstract=null), Reference(id=1241786731696029952, tenantId=1146029695717560320, journalId=1205117023404326918, articleId=1241779359137206410, doi=null, pmid=null, pmcid=null, year=2016, volume=36, issue=9, pageStart=1611, pageEnd=null, url=null, language=null, rfNumber=[19], rfOrder=21, authorNames=李谦, 刘杰, 过立农, journalName=药物分析杂志, refType=null, unstructuredReference=李谦，刘杰，过立农，等.基于ITS2序列的紫草PCR-RFLP鉴别研究[J].药物分析杂志，2016，36(9)：1611, articleTitle=基于ITS2序列的紫草PCR-RFLP鉴别研究, refAbstract=null), Reference(id=1241786731775721730, tenantId=1146029695717560320, journalId=1205117023404326918, articleId=1241779359137206410, doi=null, pmid=null, pmcid=null, year=2016, volume=36, issue=9, pageStart=1611, pageEnd=null, url=null, language=null, rfNumber=[19], rfOrder=22, authorNames=LI Q, LIU J, GUO LN, journalName=Chin J Pharm Anal, refType=null, unstructuredReference=LI Q，LIU J，GUO LN，et al. 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M.DNA Marker，由上至下分别为1 000、700、500、300、200、100 bp（from top to bottom are 1 000，700，500，300，200，100 bp）　A1~A7、AH3、B1~B5、BH3、C1~C3、CH3、D2、D5、DH3、E1~E5、EH2、F1~F5、FH3、G1~G5、GH3.进口紫草样品（imported Arnebiae Radix）　DZ.紫草对照药材（Arnebiae Radix reference drug）　K.阴性对照（negative reference）

, figureFileSmall=3H9FnkN4Vw5KYXj/OF3p4g==, figureFileBig=JnlNMHuQiwlvhCVUmYM1Xw==, tableContent=null), ArticleFig(id=1241786727791132894, tenantId=1146029695717560320, journalId=1205117023404326918, articleId=1241779359137206410, language=EN, label=Fig.2, caption=Establish the phylogenetic tree of the ITS2 sequences from imported Arnebiae Radix and Arnebiae Radix reference drug based on NJ method, figureFileSmall=k5kMTq2WOVhc7MxfNwLyJA==, figureFileBig=Y2BIXFiBSi9bK0iedYtz1w==, tableContent=null), ArticleFig(id=1241786727845658847, tenantId=1146029695717560320, journalId=1205117023404326918, articleId=1241779359137206410, language=CN, label=图2, caption=基于邻接法建立进口紫草样品与紫草对照药材ITS2序列的聚类树, figureFileSmall=k5kMTq2WOVhc7MxfNwLyJA==, figureFileBig=Y2BIXFiBSi9bK0iedYtz1w==, tableContent=null), ArticleFig(id=1241786727916962016, tenantId=1146029695717560320, journalId=1205117023404326918, articleId=1241779359137206410, language=EN, label=Tab.1, caption=

The variation point information of imported Arnebiae Radix and Arnebiae Radix reference drug

, figureFileSmall=null, figureFileBig=null, tableContent=
编号
（No.）		碱基位点（nucleotide site）
23142223262729638494133143147151153158167168180181189196200203213
121430-201705、F2ACCAAAAGGCGCCTAGCGTCGTCCTT
A5···············A··········
A1、A2、B2、C1、D2、F1、FH3、G4、G5、GH3······C········A··········
C2、C3、CH3、D5、E1、E4············T··A··········
BH3·····TT········A··········
B1、B3、B4、B5、F5·····TT········A···T··T···
A3、A4、A6、E2、E5、F4、G1、G2··A···CCC······AT····CT···
E3··A···CCC······AT···ACT···
AH3、EH2······CCC······AT····CT···
A7、G3······C······G·A··A·······
F3······C······G·A··········
DH3GA·TG·CC·TTT··GA·C·····TCC
), ArticleFig(id=1241786728013431009, tenantId=1146029695717560320, journalId=1205117023404326918, articleId=1241779359137206410, language=CN, label=表1, caption=

进口紫草与紫草对照药材的变异位点信息

, figureFileSmall=null, figureFileBig=null, tableContent=
编号
（No.）		碱基位点（nucleotide site）
23142223262729638494133143147151153158167168180181189196200203213
121430-201705、F2ACCAAAAGGCGCCTAGCGTCGTCCTT
A5···············A··········
A1、A2、B2、C1、D2、F1、FH3、G4、G5、GH3······C········A··········
C2、C3、CH3、D5、E1、E4············T··A··········
BH3·····TT········A··········
B1、B3、B4、B5、F5·····TT········A···T··T···
A3、A4、A6、E2、E5、F4、G1、G2··A···CCC······AT····CT···
E3··A···CCC······AT···ACT···
AH3、EH2······CCC······AT····CT···
A7、G3······C······G·A··A·······
F3······C······G·A··········
DH3GA·TG·CC·TTT··GA·C·····TCC
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基于DNA条形码和PCR-RFLP技术的进口紫草ITS2序列特征研究*
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刘杰 1 , 戴胜云 1 , 谷海媛 1, 2 , 乔菲 1 , 连超杰 1 , 过立农 1 , 郑健 1, ** , 马双成 1, ** , 米加 3
药物分析杂志 | 紫草类民族药材的品质评价研究专栏 2024,44(5): 750-755
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药物分析杂志 | 紫草类民族药材的品质评价研究专栏 2024, 44(5): 750-755
基于DNA条形码和PCR-RFLP技术的进口紫草ITS2序列特征研究*
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刘杰1 , 戴胜云1, 谷海媛1, 2, 乔菲1, 连超杰1, 过立农1, 郑健1, ** , 马双成1, ** , 米加3
作者信息
  • 1.中国食品药品检定研究院，北京 102629
  • 2.沈阳药科大学，沈阳 110016
  • 3.伊犁哈萨克自治州检验检测认证研究院，伊犁 835000

    • Tel：（010）53851401；E-mail：

通讯作者:

** 郑健　Tel：（010）53852080；E-mail：;
马双成　Tel：（010）53832076；E-mail：
The ITS2 sequences’ characters study of imported Arnebiae Radix based on DNA barcoding and PCR-RFLP technologies*
Jie LIU1 , Sheng-yun DAI1, Hai-yuan GU1, 2, Fei QIAO1, Chao-jie LIAN1, Li-nong GUO1, Jian ZHENG1, ** , Shuang-cheng MA1, ** , Jia MI3
Affiliations
  • 1.National Institutes for Food and Drug Control, Beijing 102629, China
  • 2.Shenyang Pharmaceutical University, Shenyang 110016, China
  • 3.Yili Institute of Inspection, Testing and Certification, Yili 835000, China
出版时间: 2024-05-31 doi: 10.16155/j.0254-1793.2024.05.02
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摘要
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目的：

基于DNA条形码和PCR-RFLP技术研究进口紫草ITS2序列的特征，为市场紫草药材和饮片的质量控制与真伪鉴别提供参考依据。

方法：

选用ITS2区域作为对进口紫草和紫草对照药材进行比较、鉴定的DNA条形码序列，并基于DNA条形码和PCR-RFLP技术比较不同来源进口紫草的ITS2序列与紫草对照药材的异同。

结果：

39份进口紫草样品经限制性内切酶AluI酶切后，其产物的琼脂糖凝胶电泳检测结果显示，仅DH3在500 bp左右有条带，而在100~300 bp无条带，其余样品均在100~300 bp有2条或3条明显条带；进口紫草样品与紫草对照药材的ITS2序列进行比对，其中样品DH3与紫草对照药材的碱基差异最多，有15个碱基差异，样品F2与紫草对照药材的ITS2序列一致，其他进口紫草样品与紫草对照药材的碱基差异为1~9个碱基；从聚类结果中可以看出进口紫草样品DH3与其他进口紫草样品和紫草对照药材均明显区分，独自为一枝，而与紫草对照药材共同聚为一枝且支持率≥50%的样品共有14个。

结论：

选用ITS2区域，基于DNA条形码和PCR-RFLP技术，比较了进口紫草与紫草对照药材ITS2序列的异同，为紫草药材及饮片的有效鉴定提供参考依据，为紫草药材的市场监管提供有力保障。

进口紫草  /  内部转录间隔区2（ITS2）  /  DNA条形码  /  限制性内切酶  /  限制性片段长度多态性聚合酶链反应
Objective:

To provide reference for quality control and authenticity identification of Arnebiae Radix medicinal materials and decoction pieces in the market. By studied on the ITS2 sequences’ characters of imported Arnebiae Radix，based on DNA barcoding and PCR-RFLP technologies.

Methods:

The ITS2 region was selected as the DNA barcode sequence for comparison and identification of imported Arnebiae Radix and reference medicinal materials. The ITS2 sequences of imported Arnebiae Radix from different sources with reference medicinal materials were compared based on DNA barcoding and PCR-RFLP technologies.

Results:

After the restriction endonucliase AluI enzyme digestion，the agarose-gel electrophoresis results of 39 imported Arnebiae Radix samples showed that，only DH3 had bands at around 500 bp，and none bands between 100 bp and 300 bp. And the results of other imported Arnebiae Radix samples had two or three obvious bands between 100 bp and 300 bp. The ITS2 sequences of imported Arnebiae Radix samples were compared with the reference medicinal materials，among which DH3 had the largest differences of 15 bases compared to the reference medicinal materials，the ITS2 sequence of F2 was same to the reference medicinal materials，and other imported Arnebiae Radix samples had 1-9 bases difference compared to the reference medicinal materials. The clustering results showed that the imported Arnebiae Radix sample DH3 was clearly distinguished from other imported Arnebiae Radix samples and reference medicinal materials which was in a single branch. There were 14 samples，which were clustered together with the reference medicinal materials in one branch with support rate ≥50%.

Conclusion:

The ITS2 region is selected to compare the similarities and differences of ITS2 sequences between imported Arnebiae Radix samples and reference medicinal materials based on DNA barcode and PCR-RFLP technologies，which provids a reference for effective identification of Arnebiae Radix medicinal materials and decoction pieces，and a strong guarantee for market supervision of Arnebiae Radix medicinal materials.

imported Arnebiae Radix  /  internal transcribed spacer 2（ITS2）  /  DNA barcoding  /  restriction endonuclease  /  polymerase chain reaction-restriction fragment length polymorphism（PCR-RFLP）
刘杰, 戴胜云, 谷海媛, 乔菲, 连超杰, 过立农, 郑健, 马双成, 米加. 基于DNA条形码和PCR-RFLP技术的进口紫草ITS2序列特征研究*. 药物分析杂志, 2024 , 44 (5) : 750 -755 . DOI: 10.16155/j.0254-1793.2024.05.02
Jie LIU, Sheng-yun DAI, Hai-yuan GU, Fei QIAO, Chao-jie LIAN, Li-nong GUO, Jian ZHENG, Shuang-cheng MA, Jia MI. The ITS2 sequences’ characters study of imported Arnebiae Radix based on DNA barcoding and PCR-RFLP technologies*[J]. Chinese Journal of Pharmaceutical Analysis, 2024 , 44 (5) : 750 -755 . DOI: 10.16155/j.0254-1793.2024.05.02
正文
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紫草因根部富含色素，最早作为古代人民印染衣物的植物染料，同时，紫草还是古代化妆品胭脂、唇脂的原料。据本草考证，紫草作为药材记载，最早见于《神农本草经》，随后历代本草中均有收载。紫草作为民族地区常用药材，同时也是常用大宗中药材，其需求量不断升高。现有可查的应用紫草药材的中成药处方122条，中药方剂195条[1]。2020年版的《中华人民共和国药典》（简称《中国药典》）中收载紫草的来源为紫草科软紫草属的新疆紫草[软紫草，Arnebia euchroma (Royle) Johnst.]、内蒙紫草（黄花软紫草，Arnebia guttata Bunge）的干燥根[2]。紫草的功能为清热凉血、活血解毒、透疹消斑。用于血热毒盛、斑疹紫黑、麻疹不透、疮疡、湿疹、水火烫伤。现代药理研究表明，紫草具有抗肿瘤、治疗皮肤病、创面修复、抗疲劳的作用[3-13]。目前市场上常见的紫草品种除2020年版《中国药典》规定的2个外，在河北、辽宁和江西等地常用硬紫草，在云南等地常用滇紫草和露蕊滇紫草，在四川等地常用密花滇紫草，在广西和广东等地常用蔷薇科的委陵菜做紫草用，俗称北紫草。由于紫草药材存在较多基原品种的问题，很多非药典规定的植物作为紫草药材混入市场，其中包括一些进口药材，导致了紫草在药材市场中的混乱情况。
中药鉴定是研究中药品种与质量，制定中药标准，寻找和扩大药源的前提和基础。近年来，中药鉴定技术的迅速发展，为中药材基原物种的鉴定奠定了良好的基础。DNA条形码（DNA barcoding）技术作为中药材物种鉴定研究中的新方法，具有鉴定结果准确、重复性良好、方法通用性强等优点[14-17]。国家药典委员会已将中药材DNA条形码分子鉴定指导原则收载于2015年版和2020年版《中国药典》四部，其在中药材鉴定、药品流通与监督等诸多方面也得到了很好的应用。ITS2区域作为中药材鉴定的潜在标准DNA条形码序列，具有可用于设计通用引物的保守区域，易于扩增，且有足够的碱基变异用以区分亲缘物种。
本试验选用ITS2区域作为对进口紫草及紫草对照药材比较、鉴定的DNA条形码序列，并基于DNA条形码和PCR-RFLP技术比较不同来源进口紫草的ITS2序列特征。
1　仪器、试药和方法
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1.1　仪器与试药
AB135-S万分之一分析天平（Mettler公司）；Milli-Q Biocel纯水仪（Millipore公司）；MM400球磨仪（Retsch公司）；ABI Veriti PCR仪（ThermoFisher公司）；EPS-301电泳仪（Amersham公司）；GelDoc XR+全自动凝胶成像系统（Biorad公司）。
植物基因组DNA提取试剂盒（批号DP305，天根生化科技有限公司）；2×Taq Master Mix缓冲液（批号GK8006，GENEray公司）；GelRed（批号41003，Biotium公司）；6×Loading buffer（货号GR0206，GENEray公司）；琼脂糖凝胶（编号A610709-0100，BBI Life Sciences）；MassRuler Express LR Forward DNA Ladder（货号SM1263，Thermo FisherTRANS）；FastDigest AluI（货号FD0014，TIANGEN）；50×TAE Buffer（货号RT204，天根生化科技有限公司）；无水乙醇（分析纯，国药基团化学试剂有限公司）；引物ITS2F：5’-ATGCGATACTTGGTGT GAAT-3’，ITS3R：5’-GACGCTTCTCCAGACTACAAT-3’引物由上海捷瑞生物工程有限公司合成。
试验收集的样品主要来源于塔吉克斯坦（编号A1~7、AH3）、吉尔吉斯斯坦（编号B1~5、BH3）、巴基斯坦（编号C1~3、CH3、F1~5、FH3、G1~5、GH3）、阿富汗（编号D2、D5、DH3）、哈萨克斯坦（编号E1~5、EH2），其中紫草对照药材（批号121430-201705）为中国食品药品检定研究院制备标定的国家标准物质。
1.2　DNA模板的制备
取紫草样品用无水乙醇常温浸泡24 h，取出晾干后用球磨仪粉碎成极细粉，平行取样每份约100 mg，利用植物基因组DNA提取试剂盒进行DNA提取，加入试剂盒中GP1后应裂解过夜（12~15 h），后续按照DNA提取试剂盒说明书进行操作。
1.3　PCR反应
PCR反应体系的制备：20 μL反应体系含2×Taq Master Mix缓冲液10 μL，上下游引物各1 μL（10 μmol·L-1），DNA模板1 μL，ddH2O 7 μL。阴性对照为无DNA模板的反应体系。PCR反应条件：以4 000 r·min-1离心10 s后，将PCR管插入PCR仪中进行反应，反应条件为95 ℃预变性4 min；95 ℃变性30 s，56 ℃退火30 s，72 ℃延伸1 min，循环35次；72 ℃延伸7 min，即得PCR扩增产物。
1.4　酶切反应
酶切反应体系的制备：30 μL反应体系含10×FastDigest 2 μL，FastDigest酶1 μL，PCR产物10 μL，ddH2O 17 μL。酶切反应条件：以4 000 r·min-1离心10 s后，将PCR管插入PCR仪中进行反应，反应条件为37 ℃酶切15 min，65 ℃灭活5 min，即得酶切产物。
1.5　琼脂糖凝胶电泳检测
1×TAE电泳缓冲液的配制：将50×TAE Buffer稀释50倍作为工作液，用于琼脂糖凝胶配制及进行凝胶电泳；2.0%琼脂糖凝胶的制备：取琼脂糖凝胶片2片（1 g），加入50 mL 1×TAE电泳缓冲液，微波炉中火加热煮沸3次至琼脂糖全部融化，摇匀，冷却至65 ℃左右加入显色剂GelRed（1：10 000）充分混匀，小心地倒入内槽板上，使胶液缓慢展开，直到整个玻璃板表面形成均匀胶层，室温下静置直至凝胶完全凝固，垂直轻拔梳子，将凝胶及内槽放入电泳槽中；加样：在已制备好的2.0%琼脂糖凝胶上用移液器将3 μL扩增产物、酶切产物、DNA Marker分别加入胶板的样品槽内；电泳：添加1×TAE电泳缓冲液至刚没过胶板为止，将加样后的凝胶板立即进行电泳，电压5 V·cm-1。当溴酚蓝（Loading buffer）移动到距离胶板下沿约2 cm处时，停止电泳；凝胶成像：用凝胶成像系统拍照并保存结果。
1.6　测序与分析
扩增产物测序：电泳检测结果显示单一明亮条带的PCR扩增产物送测序公司进行测序。测序结果分析：测序结果使用CodonCode Aligner 3.7.1软件(CodonCode Co.，USA)校对拼接，去除引物和5.8S、28S区段。将所得序列利用MEGA 6.0软件（molecular evolutionary genetics analysis）与新疆紫草对照药材的序列进行分析比对，并基于邻接法（neighbor-joining，NJ）建立聚类树，通过bootstrap（1 000次重复）对各分支进行支持率检验。
2　结果
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2.1　PCR-RFLP结果
进口紫草样品与紫草对照药材酶切产物的琼脂糖凝胶电泳检测结果如图1所示。紫草对照药材在100~300 bp有2条明显条带，300~500 bp无明显条带，阴性对照在100~300 bp和300~500 bp均无条带。39份进口紫草的酶切反应产物琼脂糖凝胶电泳检测结果显示，A~6、E1~5、F4、G1~2共12份进口紫草样品与紫草对照药材的检测结果一致，在100~300 bp有2条明显条带，300~500 bp无明显条带；A1~2、A7、B3~5、C2~3、D2、D5、F1~3、F5、G3~5共19份进口紫草样品在100~300 bp有2条明显条带，且在300~500 bp有明显条带；AH3、BH3、C1、CH3、EH2、FH3、GH3共7份进口紫草样品在100~300 bp有3条明显条带，且在300~500 bp有明显条带；DH3在500 bp左右有条带，而100~300 bp无条带。结果发现，39份进口紫草的酶切反应产物中，仅DH3不能被限制性内切酶AluI进行酶切，其余均可。限制性内切酶AluI的酶切位点为“AG^CT”，因此可以确定DH3的ITS2序列中不存在“AG^CT”位点，其余进口紫草样品均存在。
2.2　进口紫草与紫草对照药材的变异位点比较分析
ITS2引物的PCR扩增产物经琼脂糖凝胶电泳检测，结果显示为单一明亮条带，将扩增产物进行测序。测序峰图使用CodonCode Aligner 3.7.1软件校对拼接，去除5.8S和28S区段，并利用MEGA 6.0软件分析比对。紫草对照药材的ITS2序列信息为：CACATCGCGTCACCCCATCCAAAATAATGTTGGATGTGGT GGATTGTGACCTCCTGTGTCTTGAGATGCAGTTGGTCGAAATTCGAGTCCGGAGCTTAGGACTTCACGAC AAGTGGTGGTTGGATAACAACTCGCGTCATGTCGTGTGCCAAGCCTCCGTGTCTCCGTAGACCCTAAGGCGC
GTGCTTTCCAACTCGTTCGTTGGGAAACCGTGCTACGACCG，进口紫草样品与紫草对照药材的ITS2序列进行比对，具体变异位点信息见表1，其中DH3与紫草对照药材的碱基差异最多，有15个碱基差异；F2与紫草对照药材的ITS2序列一致；其他进口紫草样品与紫草对照药材的碱基差异为1~9个碱基。
2.3　邻接法聚类分析
利用MEGA 6.0软件将进口紫草样品与紫草对照药材的ITS2序列基于邻接法建立聚类树，通过bootstrap（1 000次重复）对各分支进行支持率检验，结果如图2所示，聚类树上数值表示自展支持率。可以看出DH3与其他进口紫草样品、紫草对照药材均明显区分，独自为1枝；F2与紫草对照药材聚为1枝，支持率为64%；E1、E4、D5、CH3、C3、C2、F2、A5与紫草对照药材共同聚为1枝，支持率为65%；BH3、B1、B3、B4、B5、F5与E1、E4、D5、CH3、C3、C2、F2、A5及紫草对照药材共同聚为1枝，支持率为50%；C1、A1、B2、F1、G4、GH3、F3、A7、G3、A2、D2、FH3、G5与紫草对照药材聚类的支持率低于50%；AH3、EH2、A3、A4、A6、E2、E5、F4、G1、E3、G2聚为1枝并区别于紫草对照药材，支持率为97%。
3　分析与讨论
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紫草DH3的ITS2序列为：CGAATCGCGTCACCCCATCCATGATACTCTTGGATGTGGTGGATTGTGACCTCCTGTGTCTTGAGATGCAGTTGGTCGAAATTTGAGTCCGGATCT
TAGGACTTCACGACAAGTGGTGGTTGGATAACAACTTGCGTCATGTCGTGTGCCGAACCTCCG
TGTCTCCCTAGACCCTAAGGCGCGTGCTTTCCAACTCGTTTGTCGGGAAACCGCGCTACGACCG，根据PCR-RFLP、变异位点比较分析和邻接法聚类分析的结果，进口紫草样品DH3与其他进口紫草样品及紫草对照药材的差异明显，为2015年市场紫草药材的主要来源品种，2015年该品种的市场占有率>50%。将以上DH3的ITS2序列输入到NCBI数据库的BLAST（Basic Local Alignment Search Tool）中进行比对，比对结果显示DH3的ITS2序列与序列KF287949.1一致性最高，序列KF287949.1为植物Arnebia benthamii的ITS2序列，由德克萨斯国际农工大学上传[18-19]。DH3的ITS2序列与植物Arnebia benthamii存在7个变异位点，而与紫草对照药材存在15个变异位点，说明DH3与植物Arnebia benthamii的亲缘关系更近。经查，Arnebia benthamii收载于巴基斯坦植物志（Flora of Pakistan）中，DH3收集自阿富汗，巴基斯坦位于阿富汗南部，与阿富汗有着漫长的国界线，推测DH3可能为植物Arnebia benthamii的变种或近缘物种。
限制性内切酶AluI的酶切位点为“AG^CT”，紫草对照药材及部分进口紫草样品的ITS2序列中存在“AG^CT”酶切位点，因此在PCR-RFLP结果中在100~300 bp有明显的酶切条带，而在邻接法聚类分析结果中，是对二者的ITS2序列（约220 bp）进行聚类。因此，部分进口紫草样品与紫草对照药材的PCR-RFLP结果一致，却在邻接法聚类分析时分布在不同的分枝中，且分枝的支持率>50%。以上2个结果的对比中可以发现，邻接法聚类分析的结果更能在ITS2序列中的差异。Chen等[20]对超过6 600份植物样本进行了ITS2序列鉴别能力的测试，这些植物样本在植物分类学中分布广泛，结果显示ITS2在种的水平上的鉴别成功率达92.7%，可作为中药材鉴定的潜在标准DNA条形码序列。在本课题前期研究中发现，采自新疆不同地区的新疆紫草其ITS2序列完全一致，与国内常见同科属近缘物种紫草、滇紫草、露蕊滇紫草、密花滇紫草、黄花滇紫草、黄花软紫草、疏花软紫草存在稳定差异，而进口紫草中存在ITS2序列与新疆紫草具有15个稳定变异位点的非药典品，在Genebank中比对与植物Arnebia benthamii更接近；同时也存在与新疆紫草的ITS2序列较为一致（变异位点1~9个）的样品，在Genebank中比对与植物Arnebia euchroma（新疆紫草）更接近。
DNA条形码和PCR-RFLP技术均为较成熟的技术，已在中药、民族药材及饮片的真伪鉴别中广泛应用，2020年版《中国药典》一部中，川贝母、霍山石斛、金钱白花蛇、乌梢蛇、蕲蛇的药材或饮片【鉴别】项下均收载了此类鉴别方法。目前，进口紫草的市场占有量大，填补了市场上紫草药材需求的空缺，但进口紫草并未被纳入药典标准中，其安全性、有效性、用量及进口途径等均有待考证。本文选用ITS2区域作为对进口紫草及紫草对照药材比较、鉴定的DNA条形码序列，并基于DNA条形码和PCR-RFLP技术比较不同来源进口紫草的ITS2序列特征，为了解目前药材市场中紫草药材的真实品种情况、样品来源情况以及质控标准建立提供参考，同时为紫草药材的市场监管提供保障。
基金
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  • *国家药品监督管理局药品监管科学体系建设重点项目“新技术新方法在中药质量控制中的应用(RS2024Z006-105)
  • 中国食品药品检定研究院学科带头人培养基金(2021X4)
  • 新疆维吾尔自治区药品监督管理局智力援疆创新拓展人才计划——“新疆特色民族药研究（以新疆紫草为例）团队”项目
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2024年第44卷第5期
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doi: 10.16155/j.0254-1793.2024.05.02
  • 接收时间：2023-04-12
  • 首发时间：2026-03-20
  • 出版时间：2024-05-31
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  • 收稿日期：2023-04-12
基金
*国家药品监督管理局药品监管科学体系建设重点项目“新技术新方法在中药质量控制中的应用(RS2024Z006-105)
中国食品药品检定研究院学科带头人培养基金(2021X4)
新疆维吾尔自治区药品监督管理局智力援疆创新拓展人才计划——“新疆特色民族药研究（以新疆紫草为例）团队”项目
作者信息
    1.中国食品药品检定研究院，北京 102629
    2.沈阳药科大学，沈阳 110016
    3.伊犁哈萨克自治州检验检测认证研究院，伊犁 835000

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马双成　Tel：（010）53832076；E-mail：
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2种不同金属材料的力学参数
科
Family		 属数
Number of
genus		 种数
Number of
species		 占总种数比例
Percentage of
total species (%)		 属
Genus		 种数
Number of
species		 占总种数比例
Percentage of total
species (%)
鹅膏菌科Amanitaceae 2 11 5.26 鹅膏菌属 Amanita 10 4.78
小菇科 Mycenaceae 2 12 5.74 丝盖伞属 Inocybe 5 2.39
多孔菌科 Polyporaceae 8 14 6.70 蜡蘑属 Laccaria 5 2.39
红菇科 Russulaceae 3 23 11.00 小皮伞属 Marasmius 6 2.87
小菇属 Mycena 11 5.26
光柄菇属 Pluteus 5 2.39
红菇属 Russula 17 8.13
栓菌属 Trametes 5 2.39
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