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Article(id=1241779359397253260, tenantId=1146029695717560320, journalId=1205117023404326918, issueId=1241779355555266850, articleNumber=null, orderNo=null, doi=10.16155/j.0254-1793.2024.05.03, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=null, receivedDate=1696176000000, receivedDateStr=2023-10-02, revisedDate=null, revisedDateStr=null, acceptedDate=null, acceptedDateStr=null, onlineDate=1773992870424, onlineDateStr=2026-03-20, pubDate=1717084800000, pubDateStr=2024-05-31, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1773992870424, onlineIssueDateStr=2026-03-20, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1773992870424, creator=13701087609, updateTime=1773992870424, 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=756, endPage=765, ext={EN=ArticleExt(id=1241779363792883906, articleId=1241779359397253260, tenantId=1146029695717560320, journalId=1205117023404326918, language=EN, title=The screening of identification primers for Arnebiae Radix based on nested PCR*, columnId=1241779356482208035, journalTitle=Chinese Journal of Pharmaceutical Analysis, columnName=Column on Quality Evaluation of Arnebiae Radix, runingTitle=null, highlight=null, articleAbstract=
Objective:

To design and screen specific primers for efficient amplification and identification of Arnebiae Radix from market based on the concept of nested PCR.

Methods:

Nested primers was designed using the software of Primer Premier 5 based on the ITS sequence of Arnebia euchroma and the ITS2 sequence of non-pharmacopoeial Arnebiae Radix. The amplification efficiency of genomic DNA by ITS2 universal primers PCR and nested PCR was compared. The genomic DNA of Arnebiae Radix was amplified directly by nested primers and was detected by agarose gel electrophoresis. The specific primers designed for Arnebiae Radix based on the fragment length and variation sites’ coverage of the amplified product was evaluated.

Results:

A total of 11 primers were selected for synthesis after the primers were designed by Primer Premier 5 software. The amplification efficiency of nested PCR was superior to ITS2 universal primers PCR in genomic DNA of Arnebiae Radix. The results of nested primers directly amplified genomic DNA of Arnebiae Radix by agarose gel electrophoresis were better than those of ITS2 primers，and showed a single band. Four pairs of primers，AE-9S/AE-2A，AE-4S/AE-10A，AE-12S/10A，AE-29S/AE-29A，were determined to be suitable for the identification of Arnebiae Radix.

Conclusion:

On the basis of DNA barcode identification and nested PCR technology，4 pairs of specific primers are identified which can be used to effectively distinguish Arnebia euchroma from the mainstreamed non-pharmacopoeial Arnebiae Radix in the medicinal materials market，providing reference for the subsequent research and development of identification methods for Arnebiae Radix and other traditional Chinese medicines.

, correspAuthors=Jian ZHENG, 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, Hai-yuan GU, Sheng-yun DAI, Fei QIAO, Chao-jie LIAN, Jian ZHENG, JIA Sha-er·SI Ha-ke), CN=ArticleExt(id=1241779365395108147, articleId=1241779359397253260, tenantId=1146029695717560320, journalId=1205117023404326918, language=CN, title=基于巢式PCR的紫草鉴别引物筛选*, 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：

基于巢式聚合酶链式反应（PCR）理念设计并筛选出可用于高效扩增与鉴别紫草市场样品真伪的特异性引物。

方法：

针对新疆紫草的ITS序列和紫草非《中华人民共和国药典》品ITS2序列，利用Primer Premier 5软件进行巢式引物设计；比较ITS2通用引物PCR和巢式PCR对紫草药材基因组DNA的扩增效率；基于巢式引物直接扩增紫草基因组DNA，并进行琼脂糖凝胶电泳检测；基于扩增产物片段长度、变异位点覆盖情况对设计的紫草药材特异性引物进行评价。

结果：

经过Primer Premier 5软件进行引物设计共选出11对引物进行合成；巢式PCR对紫草药材基因组DNA的扩增效率明显优于ITS2通用引物PCR；基于巢式引物直接扩增紫草基因组DNA琼脂糖凝胶电泳检测的结果明显优于ITS2引物直接扩增紫草基因组DNA，且呈单一条带；确定AE-9S/AE-2A、AE-4S/AE-10A、AE-12S/10A、AE-29S/AE-29A共4对引物适用于紫草正伪品鉴定。

结论：

在DNA条形码鉴定和巢式PCR技术的基础上，确定了4对可以用于有效区分药材市场中主流的新疆紫草和紫草非《中华人民共和国药典》品的特异性引物，为后续紫草药材及其他中药民族药品种的鉴定方法研究与开发提供了可参考的依据。

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

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Tel：（010）53851401；E-mail：

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Vet Sci China，2019，49(2)：183, articleTitle=Development of a nested PCR assay for detection of Babesia bovis, refAbstract=null), Reference(id=1241786732237099991, tenantId=1146029695717560320, journalId=1205117023404326918, articleId=1241779359397253260, doi=null, pmid=null, pmcid=null, year=2018, volume=31, issue=10, pageStart=2202, pageEnd=null, url=null, language=null, rfNumber=[14], rfOrder=24, authorNames=孙俊丽, 廖海洪, 张冰, journalName=西南农业学报, refType=null, unstructuredReference=孙俊丽，廖海洪，张冰，等.牙釉质基因巢式PCR鉴定猪ICSI胚胎性别[J].西南农业学报，2018，31(10)：2202, articleTitle=牙釉质基因巢式PCR鉴定猪ICSI胚胎性别, refAbstract=null), Reference(id=1241786732312597466, tenantId=1146029695717560320, journalId=1205117023404326918, articleId=1241779359397253260, doi=null, pmid=null, pmcid=null, year=2018, volume=31, issue=10, pageStart=2202, pageEnd=null, url=null, language=null, rfNumber=[14], rfOrder=25, authorNames=SUN JL, LIAO HH, ZHANG B, journalName=Southwest China J Agric Sci, refType=null, unstructuredReference=SUN JL，LIAO HH，ZHANG B，et al. Sex identification of porcine ICSI embryo by nested amplification of amelogenin gene[J]. Southwest China J Agric Sci，2018，31(10)：2202, articleTitle=Sex identification of porcine ICSI embryo by nested amplification of amelogenin gene, refAbstract=null), Reference(id=1241786732400677852, tenantId=1146029695717560320, journalId=1205117023404326918, articleId=1241779359397253260, doi=null, pmid=null, pmcid=null, year=2018, volume=29, issue=5, pageStart=425, pageEnd=null, url=null, language=null, rfNumber=[15], rfOrder=26, authorNames=张琳, 苗广青, 侯学霞, journalName=中国媒介生物学及控制杂志, refType=null, unstructuredReference=张琳，苗广青，侯学霞，等.巢式PCR和实时荧光定量PCR在莱姆病宿主动物监测中的应用评价[J].中国媒介生物学及控制杂志，2018，29(5)：425, articleTitle=巢式PCR和实时荧光定量PCR在莱姆病宿主动物监测中的应用评价, refAbstract=null), Reference(id=1241786732505535457, tenantId=1146029695717560320, journalId=1205117023404326918, articleId=1241779359397253260, doi=null, pmid=null, pmcid=null, year=2018, volume=29, issue=5, pageStart=425, pageEnd=null, url=null, language=null, rfNumber=[15], rfOrder=27, authorNames=ZHANG L, MIAO GQ, HOU XX, journalName=Chin J Vector Biol Control, refType=null, unstructuredReference=ZHANG L，MIAO GQ，HOU XX，et al. Evaluation of nested PCR and real-time PCR in host surveillance of Lyme disease[J]. 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M.marker　1~25.实验样品（experimental samples）

, figureFileSmall=WrhkTI+btZ30HNndVPw4dw==, figureFileBig=4mSi+heFAGzZi6lmzGRSpQ==, tableContent=null), ArticleFig(id=1241786727610782573, tenantId=1146029695717560320, journalId=1205117023404326918, articleId=1241779359397253260, language=EN, label=Fig.2, caption=The agarose gel electrophoresis result of polymerase chain reaction after optimized reaction procedures, figureFileSmall=U9+DTzvekHVgGagVXAQ61Q==, figureFileBig=xwEPsFfERdrjW/om893yQg==, tableContent=null), ArticleFig(id=1241786727703057263, tenantId=1146029695717560320, journalId=1205117023404326918, articleId=1241779359397253260, language=CN, label=图2, caption=优化反应程序后的PCR琼脂糖凝胶电泳检测结果

M.marker　1~25.实验样品（experimental samples）

, figureFileSmall=U9+DTzvekHVgGagVXAQ61Q==, figureFileBig=xwEPsFfERdrjW/om893yQg==, tableContent=null), ArticleFig(id=1241786727757583216, tenantId=1146029695717560320, journalId=1205117023404326918, articleId=1241779359397253260, language=EN, label=Fig.3, caption=The agarose gel electrophoresis results of nPCR, figureFileSmall=Pe4CBv44IfirAUaZNBFHFw==, figureFileBig=CxBy2rjIAENzvZXHVJlk5g==, tableContent=null), ArticleFig(id=1241786727816303473, tenantId=1146029695717560320, journalId=1205117023404326918, articleId=1241779359397253260, language=CN, label=图3, caption=巢式PCR的琼脂糖凝胶电泳结果

M.marker　1~25.实验样品（experimental samples）

, figureFileSmall=Pe4CBv44IfirAUaZNBFHFw==, figureFileBig=CxBy2rjIAENzvZXHVJlk5g==, tableContent=null), ArticleFig(id=1241786727895995250, tenantId=1146029695717560320, journalId=1205117023404326918, articleId=1241779359397253260, language=EN, label=Fig.4, caption=The agarose gel electrophoresis results of Arnebiae Radix genomic DNA directly amplified by nPCR primers, figureFileSmall=GZgVVaqP2fwwflG0kLGG0w==, figureFileBig=swdk6te6R2FiqZ/sf8RQxQ==, tableContent=null), ArticleFig(id=1241786727992464243, tenantId=1146029695717560320, journalId=1205117023404326918, articleId=1241779359397253260, language=CN, label=图4, caption=巢式引物直接扩增紫草基因组DNA的琼脂糖凝胶电泳结果

M.marker　1~25.实验样品（experimental samples）

, figureFileSmall=GZgVVaqP2fwwflG0kLGG0w==, figureFileBig=swdk6te6R2FiqZ/sf8RQxQ==, tableContent=null), ArticleFig(id=1241786728051184500, tenantId=1146029695717560320, journalId=1205117023404326918, articleId=1241779359397253260, language=EN, label=Tab.1, caption=

The primers’ results designed based on the nPCR concept

, figureFileSmall=null, figureFileBig=null, tableContent=
序号（No.）引物名称（primer name）引物序列5’-3’（primer sequences 5’-3’）退火温度（annealing temperature）/℃
1AE-2SAATAATGTTGGATGTGGTGGAT50
AE-2AGGTCGTAGCACGGTTTCC
2AE-4SGTTGGATGTGGTGGATTG48
AE-4AGACATGACGCGAGTTGTT
3AE-5SGACAAGTGGTGGTTGGATA49
AE-2AGGTCGTAGCACGGTTTCC
4AE-7SCCATTAGGCTAAAGGCACG50
AE-7ACACGGTTTCCCAACGAAC
5AE-9SCAAGTTGCGCCTGAGGCCATTA55
AE-2AGGTCGTAGCACGGTTTCC
6AE-4SGTTGGATGTGGTGGATTG48
AE-10ACCTCCGACTTATTGATATGCT
7AE-12STGGATGTGGTGGATTGTG48
AE-10ACCTCCGACTTATTGATATGCT
8AE-6SCGAAATGCGATACTTGGTG50
AE-6AGTAATCCCGCCTGACCTG
9AE-29STGAAGAACGTAGCGAAATG52
AE-29AACACGGAGGCTTGGCACA
10ZCF02918CTTGGATGTGGTGGATTG52
ZCF16918TAGGGAGACACGGAGGTT
11ZCF03118TGGATGTGGTGGATTGTG52
ZCF18019GCCTTAGGGTCTAGGGAGA
), ArticleFig(id=1241786728126681973, tenantId=1146029695717560320, journalId=1205117023404326918, articleId=1241779359397253260, language=CN, label=表1, caption=

基于巢式PCR理念的引物设计结果

, figureFileSmall=null, figureFileBig=null, tableContent=
序号（No.）引物名称（primer name）引物序列5’-3’（primer sequences 5’-3’）退火温度（annealing temperature）/℃
1AE-2SAATAATGTTGGATGTGGTGGAT50
AE-2AGGTCGTAGCACGGTTTCC
2AE-4SGTTGGATGTGGTGGATTG48
AE-4AGACATGACGCGAGTTGTT
3AE-5SGACAAGTGGTGGTTGGATA49
AE-2AGGTCGTAGCACGGTTTCC
4AE-7SCCATTAGGCTAAAGGCACG50
AE-7ACACGGTTTCCCAACGAAC
5AE-9SCAAGTTGCGCCTGAGGCCATTA55
AE-2AGGTCGTAGCACGGTTTCC
6AE-4SGTTGGATGTGGTGGATTG48
AE-10ACCTCCGACTTATTGATATGCT
7AE-12STGGATGTGGTGGATTGTG48
AE-10ACCTCCGACTTATTGATATGCT
8AE-6SCGAAATGCGATACTTGGTG50
AE-6AGTAATCCCGCCTGACCTG
9AE-29STGAAGAACGTAGCGAAATG52
AE-29AACACGGAGGCTTGGCACA
10ZCF02918CTTGGATGTGGTGGATTG52
ZCF16918TAGGGAGACACGGAGGTT
11ZCF03118TGGATGTGGTGGATTGTG52
ZCF18019GCCTTAGGGTCTAGGGAGA
), ArticleFig(id=1241786728181207927, tenantId=1146029695717560320, journalId=1205117023404326918, articleId=1241779359397253260, language=EN, label=Tab.2, caption=

The evaluation information table of nested primers

, figureFileSmall=null, figureFileBig=null, tableContent=
序号
（No.）		引物名称
（primer name）		引物位于新疆紫草ITS序列中的位置
（primer location in ITS sequence of Arnebia euchroma）		扩增产物片段大小（扩增产物变异位点数）
[fragment size of amplified product（variants number of amplified product）]
1AE-2S428-449~200 bp(8)
AE-2A610-627
2AE-4S434-451100~200 bp(/)
AE-4A531-548
3AE-5S513-531100~200 bp(/)
AE-2A610-627
4AE-7S372-390200~300 bp(12)
AE-7A602-619
5AE-9S356-377200~300 bp(14)
AE-2A610-627
6AE-4S434-451200~300 bp(11)
AE-10A665-685
7AE-12S436-453200~300 bp(11)
AE-10A665-685
8AE-6S298-316300~400 bp(/)
AE-6A635-652
9AE-29S286-304~300 bp(9)
AE-29A550-567
10ZCF02918434-451100~200 bp(/)
ZCF16918557-574
11ZCF03118436-453100~200 bp(/)
ZCF18019567-585
), ArticleFig(id=1241786728260899703, tenantId=1146029695717560320, journalId=1205117023404326918, articleId=1241779359397253260, language=CN, label=表2, caption=

巢式引物评价信息表

, figureFileSmall=null, figureFileBig=null, tableContent=
序号
（No.）		引物名称
（primer name）		引物位于新疆紫草ITS序列中的位置
（primer location in ITS sequence of Arnebia euchroma）		扩增产物片段大小（扩增产物变异位点数）
[fragment size of amplified product（variants number of amplified product）]
1AE-2S428-449~200 bp(8)
AE-2A610-627
2AE-4S434-451100~200 bp(/)
AE-4A531-548
3AE-5S513-531100~200 bp(/)
AE-2A610-627
4AE-7S372-390200~300 bp(12)
AE-7A602-619
5AE-9S356-377200~300 bp(14)
AE-2A610-627
6AE-4S434-451200~300 bp(11)
AE-10A665-685
7AE-12S436-453200~300 bp(11)
AE-10A665-685
8AE-6S298-316300~400 bp(/)
AE-6A635-652
9AE-29S286-304~300 bp(9)
AE-29A550-567
10ZCF02918434-451100~200 bp(/)
ZCF16918557-574
11ZCF03118436-453100~200 bp(/)
ZCF18019567-585
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基于巢式PCR的紫草鉴别引物筛选*
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刘杰 1 , 谷海媛 1, 2 , 戴胜云 1 , 乔菲 1 , 连超杰 1 , 郑健 1, ** , 加沙尔·斯哈克 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): 756-765
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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): 756-765
基于巢式PCR的紫草鉴别引物筛选*
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刘杰1 , 谷海媛1, 2, 戴胜云1, 乔菲1, 连超杰1, 郑健1, ** , 加沙尔·斯哈克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:

** Tel：（010）53852080；E-mail：
The screening of identification primers for Arnebiae Radix based on nested PCR*
Jie LIU1 , Hai-yuan GU1, 2, Sheng-yun DAI1, Fei QIAO1, Chao-jie LIAN1, Jian ZHENG1, ** , JIA Sha-er·SI Ha-ke3
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.0254-1793.2024.05.03
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摘要
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目的：

基于巢式聚合酶链式反应（PCR）理念设计并筛选出可用于高效扩增与鉴别紫草市场样品真伪的特异性引物。

方法：

针对新疆紫草的ITS序列和紫草非《中华人民共和国药典》品ITS2序列，利用Primer Premier 5软件进行巢式引物设计；比较ITS2通用引物PCR和巢式PCR对紫草药材基因组DNA的扩增效率；基于巢式引物直接扩增紫草基因组DNA，并进行琼脂糖凝胶电泳检测；基于扩增产物片段长度、变异位点覆盖情况对设计的紫草药材特异性引物进行评价。

结果：

经过Primer Premier 5软件进行引物设计共选出11对引物进行合成；巢式PCR对紫草药材基因组DNA的扩增效率明显优于ITS2通用引物PCR；基于巢式引物直接扩增紫草基因组DNA琼脂糖凝胶电泳检测的结果明显优于ITS2引物直接扩增紫草基因组DNA，且呈单一条带；确定AE-9S/AE-2A、AE-4S/AE-10A、AE-12S/10A、AE-29S/AE-29A共4对引物适用于紫草正伪品鉴定。

结论：

在DNA条形码鉴定和巢式PCR技术的基础上，确定了4对可以用于有效区分药材市场中主流的新疆紫草和紫草非《中华人民共和国药典》品的特异性引物，为后续紫草药材及其他中药民族药品种的鉴定方法研究与开发提供了可参考的依据。

紫草  /  聚合酶链式反应(PCR)  /  内部转录间隔区2(ITS2)  /  DNA条形码  /  巢式PCR(nPCR)
Objective:

To design and screen specific primers for efficient amplification and identification of Arnebiae Radix from market based on the concept of nested PCR.

Methods:

Nested primers was designed using the software of Primer Premier 5 based on the ITS sequence of Arnebia euchroma and the ITS2 sequence of non-pharmacopoeial Arnebiae Radix. The amplification efficiency of genomic DNA by ITS2 universal primers PCR and nested PCR was compared. The genomic DNA of Arnebiae Radix was amplified directly by nested primers and was detected by agarose gel electrophoresis. The specific primers designed for Arnebiae Radix based on the fragment length and variation sites’ coverage of the amplified product was evaluated.

Results:

A total of 11 primers were selected for synthesis after the primers were designed by Primer Premier 5 software. The amplification efficiency of nested PCR was superior to ITS2 universal primers PCR in genomic DNA of Arnebiae Radix. The results of nested primers directly amplified genomic DNA of Arnebiae Radix by agarose gel electrophoresis were better than those of ITS2 primers，and showed a single band. Four pairs of primers，AE-9S/AE-2A，AE-4S/AE-10A，AE-12S/10A，AE-29S/AE-29A，were determined to be suitable for the identification of Arnebiae Radix.

Conclusion:

On the basis of DNA barcode identification and nested PCR technology，4 pairs of specific primers are identified which can be used to effectively distinguish Arnebia euchroma from the mainstreamed non-pharmacopoeial Arnebiae Radix in the medicinal materials market，providing reference for the subsequent research and development of identification methods for Arnebiae Radix and other traditional Chinese medicines.

Arnebiae Radix  /  polymerase chain reaction (PCR)  /  internal transcribed spacer Ⅱ of nuclear ribosomal DNA (ITS2)  /  DNA barcoding  /  nested polymerase chain reaction (nPCR)
刘杰, 谷海媛, 戴胜云, 乔菲, 连超杰, 郑健, 加沙尔·斯哈克. 基于巢式PCR的紫草鉴别引物筛选*. 药物分析杂志, 2024 , 44 (5) : 756 -765 . DOI: 10.16155/j.0254-1793.2024.05.03
Jie LIU, Hai-yuan GU, Sheng-yun DAI, Fei QIAO, Chao-jie LIAN, Jian ZHENG, JIA Sha-er·SI Ha-ke. The screening of identification primers for Arnebiae Radix based on nested PCR*[J]. Chinese Journal of Pharmaceutical Analysis, 2024 , 44 (5) : 756 -765 . DOI: 10.16155/j.0254-1793.2024.05.03
正文
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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条形码技术作为新的分子生物学物种鉴定手段，具有鉴定结果准确，重复性良好，方法通用性强等优点[1，2]，在传统形态分类学对物种准确鉴定的基础上，通过对样品标准序列进行测序，建立DNA条形码数据库，可以实现物种鉴定的标准化。国家药典委员会自2015年版《中华人民共和国药典》（简称《中国药典》）开始收载了中药材DNA条形码分子鉴定指导原则，促进了DNA条形码技术在中药材鉴定、药品流通与监督等诸多方面的应用[2-5]。
聚合酶链式反应（polymerase chain reaction，PCR）借助PCR仪按照相应的时间和循环次数对靶基因序列进行扩增，由变性、退火和延伸构成1个完整的反应周期。常规PCR具有程序简单、省时等优点，但常规PCR在退火温度不适当时会出现假阳性，而巢式PCR（nested polymerase chain reaction，nPCR）通过使用内、外2对PCR引物分别进行扩增而显著提升了PCR的敏感性和准确性。
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，具有足够的变异用以区分亲缘物种。ITS区域包含ITS2区域，具有更长的序列长度和更多的变异位点数。为针对紫草建立较为专属的巢式PCR鉴别方法，考虑针对紫草的ITS和ITS2区域设计巢式特异性引物。通过琼脂糖凝胶电泳的方法对特异性引物的巢式PCR扩增效果进行评价，筛选出扩增效果好，专属性强的特异性引物。
2020年版的《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。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，由于国内新疆紫草的产量难以满足市场需求，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，经过对全国市场的紫草药材抽验发现，市场上主要流通的紫草药材为新疆紫草和非药典收载的进口紫草品种（非药典品），没有发现内蒙紫草作为紫草药材在市场上流通。紫草药材的市场样品因受保存时间长、保存条件差、次生代谢产物含量高等原因的限制，导致其基因组DNA降解严重。药材的基因组DNA提取和PCR扩增的有效性是保证药材能够进行分子生物学鉴定的基础与前提，针对紫草药材的特殊性，对其PCR扩增条件进行优化，可提高紫草市场药材DNA条形码短片段的PCR扩增成功率，解决紫草药材市场来源样品进行DNA条形码鉴定的前端问题，为中药、民族药市场药材的成功鉴定奠定基础。
本试验拟针对紫草的ITS和ITS2区域设计并筛选巢式特异性扩增引物，为紫草市场药材的成功鉴定提供支撑。
1　材料和方法
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1.1　材料
AB135-S万分之一分析天平（Mettler公司），MM400球磨仪（Retsch公司），NanoDrop one超微量分光光度计（Therno Fisher公司），ABI Veriti PCR仪（ThernoFisher公司），Mupid-one水平电泳仪（Takara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（货号DP305，天根生化科技有限公司），2×Taq Master Mix缓冲液（货号GK8006，GENEray公司），GelRed（货号41003，Biotium公司），Fast Dissolved Agarose Tablets（货号A610709-0100，BBI Life Sciences），Trans DNA Marker Ⅰ（货号BM401，TransGen Biotech公司），50×TAE Buffer（货号RT204，天根生化科技有限公司）。引物ITS2（ITS2F：5’-ATGCGATACTTGGTGTGAAT-3’，ITS3R：5’-GACGCTTCTCCA GACTACAAT-3’），引物ITS（ITS4：5’-TCCTCCGCTTATTGATATGC-3’，ITS5：5’- GGAAGTAAAAGTCGTAACAAGG-3’）（上海捷瑞生物工程有限公司）。
实验共选取25批紫草样品，通过全国药材抽验收集自药材生产或经营单位，经DNA条形码鉴定其来源并由中国食品药品检定研究院郑健研究员审核。
1.2　植物总DNA的提取
利用植物基因组提取试剂盒对紫草样品进行基因组DNA提取，首先将紫草样品用100%乙醇常温浸泡24 h，取出晾干后经MM400进行粉碎，取样量约为100 mg，加入植物基因组提取试剂盒中的GP1后裂解过夜（12~15 h），后续操作按照植物基因组DNA提取试剂盒的操作步骤进行基因组DNA提取。
1.3　基于ITS、ITS2引物的PCR
PCR反应体系20 μL：2×Taq Master Mix缓冲液10 μL，上下游引物各0.4 μL（2.5 μmol·L-1），DNA模板1 μL，灭菌的双蒸水8.6 μL。PCR反应条件：95 ℃预变性4 min；95 ℃变性30 s，52 ℃退火30 s，72 ℃延伸1 min，循环35次；72 ℃延伸7 min。PCR优化扩增程序：95 ℃预变性4 min；95 ℃变性30 s，40 ℃退火30 s，72 ℃延伸1 min，循环5次；95 ℃变性30 s，52 ℃退火30 s，72 ℃延伸1 min，循环35次；72 ℃延伸7 min。
1.4　基于巢式PCR理念的引物设计
ITS引物扩增新疆紫草的基因组DNA，得到的ITS序列去除引物端，ITS2引物扩增紫草市场非药典品的基因组DNA，得到的ITS2序列去除5.8S和28S区段。利用Primer Premier 5软件针对以上新疆紫草的ITS序列和紫草市场非药典品的ITS2序列设计引物。
1.5　基于巢式引物的聚合酶链式反应
反应体系20 μL：2×Taq Master Mix缓冲液10 μL，上下游引物各0.4 μL（2.5 μmol·L-1），模板（PCR扩增产物或基因组DNA）1 μL，灭菌的双蒸水8.6 μL。PCR反应条件：95 ℃预变性4 min；95 ℃变性30 s，52 ℃退火30 s，72 ℃延伸1 min，循环35次；72 ℃延伸7 min。PCR优化扩增程序：95 ℃预变性4 min；95 ℃变性30 s，40 ℃退火30 s，72 ℃延伸1 min，循环5次；95 ℃变性30 s，52 ℃退火30 s，72 ℃延伸1 min，循环35次；72 ℃延伸7 min。
1.6　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，添加1×TAE电泳缓冲液至刚没过胶板为止。加样：在在配制好的琼脂糖凝胶板上将3 μL扩增产物、DNA Marker用移液器分别将样品加入胶板的样品槽内。电泳：加样后的凝胶板立即进行电泳，电压5 V·cm-1。凝胶成像：用凝胶成像系统拍照并保存结果。
2　结果
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2.1　基于ITS2引物的聚合酶链式反应电泳检测结果
以ITS2引物对紫草样品的基因组DNA进行PCR扩增，扩增产物经琼脂糖凝胶电泳检测，结果如图1所示，可能受基因组DNA降解程度或DNA中的杂质干扰扩增酶活性的影响，扩增结果中存在较多未扩出或扩增产物条带不明显的样品。为改善ITS2引物对紫草基因组DNA的PCR扩增效果，对其PCR扩增程序进行调整，在进行95 ℃预变性4 min的反应后，增加95 ℃变性30 s、40 ℃退火30 s、72 ℃延伸1 min的反应循环5次，然后再进行95 ℃变性30 s，52 ℃退火30 s，72 ℃延伸1 min，循环35次，其扩增产物经琼脂糖凝胶电泳检测，结果如图2所示，扩增效果并没有达到预期中惊艳的效果，扩增结果中存在较多为扩出或扩增产物条带不明显的样品。因此，考虑通过巢式PCR的方法，改善紫草样品基因组DNA的扩增效果，为后续鉴定奠定基础。
2.2　基于巢式PCR理念的引物设计结果
经DNA条形码鉴定，市场抽样的紫草样品主要有新疆紫草和紫草市场非药典品，新疆紫草的ITS序列和紫草市场非药典品的ITS2序列如下所示：
新疆紫草（Arnebia euchroma）-ACCGGGT-TTATCTTGTCGATCCTGCACAGCAGAACCACTCGCGAACAAGTTCTCAAAACCAAGGCACCGGTCGGTGCAAGGAACACCTTGCTCT
CGATCGGAGCCCAACGGTGCCACGACATCTTGTCGTGGCGCTGAACAAACCCCCGGCGCGAGAAGCGCCAAGGAATACTTTAACGAGAGCTT
GAACCATCCTCTCCCGTTCGCGGGGCTTAGGGGCTTGGAGACGGCTTCTTTATAAACCACAACGACTCTCGGCAACGGATATCTTGGCTCTCGC
ATCGATGAAGAACGTAGCGAAATGCGATACTTGGTGTGAATTGCAGAATCCCGTGAACCATCGAGTCTTTGAACGCAAGTTGCGCCTGAGGCCA
TTAGGCTAAAGGCACGTCTGCCTGGGCGTCACACATCGCGTCACCCCATCCAAAATAATGTTGGATGTGGTGGATTGTGACCTCCTGTGTCTTG
AGATGCAGTTGGTCGAAATTCGAGTCCGGAGCTTAGGACTTCACGACAAGTGGTGGTTGGATAACAACTCGCGTCATGTCGTGTGCCAAGCCTC
CGTGTCTCCGTAGACCCTAAGGCGCGTGCTTTCCAACTCGTTCGTTGGGAAACCGTGCTACGACCGCGACCCCAGGTCAGGCGGGATTACCCGC
TGAATTTAAGCATATCAATAAGTCGGAGGAT
紫草市场非药典品-CGAATCGCGTCACCCCATCCATGATACTCTTGGATGTGGTGGAT TGTGACCTCCTGTGTCTTGA
GATGCAGTTGGTCGAAATTTGAGTCCGGATCTTAGGACTTCACGACAAGTGGTGGTTGGATAACAACTTGCGTCATGTCGTGTGCCGAACCTC
CGTGTCTCCCTAGACCCTAAGGCGCGTGCTTTCCAACTCGTTTGTCGGGAAACCGCGCTACGACCGCCG
将以上新疆紫草和紫草市场非药典品的序列输入到Primer Premier 5软件中，综合考虑所得引物序列的评分和适用性，共筛选出11对引物，由捷瑞公司合成引物。引物详细信息如表1所示。
2.3　巢式PCR的琼脂糖凝胶电泳结果
将通用引物扩增紫草基因组DNA的PCR产物作为模板，分别以表1中的11对引物作为反应引物进行巢式PCR扩增，结果如图3所示，紫草基因组DNA经巢式PCR两步扩增后，其扩增成功率基本接近100%，具有很好的扩增效果，扩增条带主要集中在100~300 bp，部分样品经巢式PCR扩增与琼脂糖凝胶电泳检测，结果呈单一明亮条带，也有部分样品呈多条带或弥散条带。
2.4　基于巢式引物直接扩增紫草基因组DNA的琼脂糖凝胶电泳结果
为了验证以巢式引物直接扩增紫草基因组DNA是否具有与巢式PCR相同的扩增效率，进一步做出实验验证，结果如图4所示，其中4对引物的扩增产物片段大小位于100~200 bp，分别为AE-4S/AE-4A、AE-5S/AE-2A、ZCF03118/ZCF18019、ZCF02918/ZCF16918；有5对引物的扩增产物片段大小位于200~300 bp，分别为AE-7S/AE-7A、AE-2S/AE-2A、AE-12S/AE-10A、AE-4S/AE-10A、AE-9S/AE-2A；有2对引物的扩增产物片段大小位于300~400 bp，分别为AE-6S/AE-6A和AE-29S/AE-29A。与前面“2.3”中2次PCR扩增得到的结果相比，扩增效率虽不能达到100%，但明显优于ITS2引物直接扩增紫草基因组DNA，且呈单一条带，这对后续紫草药材鉴别中的测序与序列比对具有重要意义。
2.5　巢式引物的评价
经DNA条形码鉴定，市场抽样的紫草样品主要有新疆紫草和紫草市场非药典品[6-7]，新疆紫草的ITS序列和紫草市场非药典品的ITS2序列如下所示：
新疆紫草（Arnebia euchroma）-ACCGGGTTTATCTTGTCGATCCTGCACAGCAGAACCACTCGCGAACAAGTTCTCAAAACCAAGGCACCGGTCGGTGCAAGGAACACC
TTGCTCTCGATCGGAGCCCAACGGTGCCACGACATCTTGTCGTGGCGCTGAACAAACCCCCGGCGCGAGAAGCGCCAAGGAA
TACTTTAACGAGAGCTTGAACCATCCTCTCCCGTTCGCGGGGCTTAGGGGCTTGGAGACGGCTTCTTTATAAACCACAACGA
CTCTCGGCAACGGATATCTTGGCTCTCGCATCGATGAAGAACGTAGCGAAATGCGATACTTGGTGTGAATTGCAGAATCCCG
TGAACCATCGAGTCTTTGAACGCAAGTTGCGCCTGAGGCCATTAGGCTAAAGGCACGTCTGCCTGGGCGTCACACATCGCGT
CACCCCATCCAAAATAATGTTGGATGTGGTGGATTGTGACCTCCTGTGTCTTGAGATGCAGTTGGTCGAAATTCGAGTCCGG
AGCTTAGGACTTCACGACAAGTGGTGGTTGGATAACAACTCGCGTCATGTCGTGTGCCAAGCCTCCGTGTCTCCGTAGACCC
TAAGGCGCGTGCTTTCCAACTCGTTCGTTGGGAAACCGTGCTACGACCGCGACCCCAGGTCAGGCGGGATTACCCGCTGAAT
TTAAGCATATCAATAAGTCGGAGGAT
紫草市场非药典品-CGAATCGCGTCACCCCATCCATGATACTCTTGGATGTGGTGGATTGTGACCTCCTGTGTCTTGAG
ATGCAGTTGGTCGAAATTTGAGTCCGGATCTTAGGACTTCACGACAAGTGGTGGTTGGATAACAACTTGCGTCATGTCGTGT
GCCGAACCTCCGTGTCTCCCTAGACCCTAAGGCGCGTGCTTTCCAACTCGTTTGTCGGGAAAACCGCGCTACGACCGCCG
以上2条序列经MEGA（Molecular Evolutionary Genetics Analysis）软件进行自动序列对齐（automatic sequence alignment）后，非药典品的ITS2序列与新疆紫草ITS序列的406~631 bp对齐，且以上2条序列对齐部分共存在17个变异位点，变异位点位置与对应变异信息：407 A-G；408 C-A；427 A-T；428 A-G；432 A-C；434 G-C；489 C-T；499 G-T；538 C-T；556 A-G；558 G-A；572 G-C；605 C-T；608 T-C；618 T-C；630 G-C；631 A-G。
综合考虑11对引物的扩增产物片段长度、变异位点覆盖情况(表2)，以及扩增产物经琼脂糖凝胶电泳检测的条带亮度，确定AE-9S/AE-2A、AE-4S/AE-10A、AE-12S/AE-10A、AE-29S/AE-29A共4对引物适用于紫草正伪品鉴定。
3　讨论与分析
收起
DNA条形码技术为药用植物鉴别开辟了1条新途径，使不能从性状上进行准确鉴定的中药、民族药样品依靠新的分子生物学手段完成快速鉴定成为可能。巢式PCR技术具有操作简单，灵敏度高，特异性强，重复性好，快速等优点，该技术是由内外2对引物先后对靶序列进行扩增，使其DNA拷贝数以双重指数级增加，常规PCR扩增对模板浓度有一定要求，微量样品的鉴定则存在一定的难度，而巢式PCR扩增的出现解决了这一困境。同时，巢式PCR扩增的第2次扩增是以第1次PCR扩增产物为模板，其引物结合到第1次PCR产物序列的内部，再加入Taq酶进行再次PCR反应，该方法既增加了模板量又保证了Taq酶的活性，如果第1次扩增发生了错误片段，则第2次能在错误片段上形成引物配对并扩增的概率很低，基本上不会出现非特异性片段。因此，与常规PCR相比，巢式PCR的灵敏度更高、特异性更强[8-15]。
本研究在DNA条形码鉴定的基础上，针对紫草药材的ITS2序列进行巢式引物设计，提高了紫草药材的扩增效率，为市场样品进行真伪鉴别奠定了重要基础，但巢式PCR产物经琼脂糖凝胶电泳检测后，其结果中有部分样品呈多条带或弥散条带，且巢式PCR经两步扩增后，其扩增效率虽然大大提高，但第二步扩增是以第一步扩增的产物作为模板，容易导致实验环境及样品的污染，影响结果鉴定的准确性。因此，虽然巢式PCR扩增的结果明显优于ITS2引物的常规PCR扩增结果，本课题组仍继续考察了舍去第一步ITS2引物扩增，选用巢式引物直接扩增紫草药材基因组DNA的琼脂糖凝胶电泳结果。结果显示，巢式引物的扩增效果优于ITS2引物，且条带单一，适用于后续的测序及鉴定。
根据扩增效率、扩增产物长度及扩增片段所包含的新疆紫草与紫草非药典品变异位点数对表1及2中11对引物进行优选，结果显示，AE-9S/AE-2A、AE-4S/AE-10A、AE-12S/AE-10A、AE-29S/AE-29A此4对引物直接扩增紫草基因组DNA的扩增效率明显优于ITS2引物，扩增产物片段长度约200~300 bp，这个片段长度低于ITS2引物的扩增片断长度，对于紫草药材降解后的基因组DNA也能保证很好的扩增效率，同时还能在琼脂糖凝胶电泳检测时与扩增过程中可能产生的引物二聚体明显区分开。新疆紫草与紫草非药典品经此4对引物扩增后，扩增产物存在9~14个变异位点，能够明显区分紫草药材正伪品，达到对市场紫草药材进行真伪鉴别的目的。
本研究在DNA条形码鉴定和巢式PCR技术的基础上，确定了4对可以用于有效区分药材市场中主流的新疆紫草和紫草非药典品的特异性引物，为后续紫草药材的鉴定方法研究与开发奠定了基础，同时为紫草药材的标准提升与补充分子生物学鉴别标准提供了可参考的依据。
基金
收起
  • *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
文献
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2024年第44卷第5期
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doi: 10.16155/j.0254-1793.2024.05.03
  • 接收时间：2023-10-02
  • 首发时间：2026-03-20
  • 出版时间：2024-05-31
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  • 收稿日期：2023-10-02
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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2种不同金属材料的力学参数
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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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