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Article(id=1240688794454782890, tenantId=1146029695717560320, journalId=1205117023404326918, issueId=1240688783964819588, articleNumber=null, orderNo=null, doi=10.16155/j.0254-1793.2024-0035, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=null, receivedDate=1705507200000, receivedDateStr=2024-01-18, revisedDate=null, revisedDateStr=null, acceptedDate=null, acceptedDateStr=null, onlineDate=1773732859481, onlineDateStr=2026-03-17, pubDate=1727625600000, pubDateStr=2024-09-30, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1773732859481, onlineIssueDateStr=2026-03-17, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1773732859481, creator=13701087609, updateTime=1773732859481, updator=13701087609, issue=Issue{id=1240688783964819588, tenantId=1146029695717560320, journalId=1205117023404326918, year='2024', volume='44', issue='9', pageStart='1463', pageEnd='1645', issueExtLink='null', onlineDate='null', pubDate='null', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=null, createTime=1773732856979, creator=13701087609, updateTime=1773733032821, updator=13701087609, preIssue=null, nextIssue=null, ext={EN=IssueExt(id=1240689521587712627, tenantId=1146029695717560320, journalId=1205117023404326918, issueId=1240688783964819588, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1240689521587712628, tenantId=1146029695717560320, journalId=1205117023404326918, issueId=1240688783964819588, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null}, startPage=1529, endPage=1534, ext={EN=ArticleExt(id=1240688794802910149, articleId=1240688794454782890, tenantId=1146029695717560320, journalId=1205117023404326918, language=EN, title=Rapid authentication of Eupolyphaga sinensis by loop-mediated isothermal amplification, columnId=1206272758774821315, journalTitle=Chinese Journal of Pharmaceutical Analysis, columnName=Standard Deliberation, runingTitle=null, highlight=null, articleAbstract=

Objective: To establish an authentication method for Eupolyphaga sinensis based on loop-mediated isothermal amplification (LAMP). Methods: Two pairs of primers （outer primer DB-F3, DB-B3 and inner primer DB-FIP, DB-BIP） were designed according to Eupolyphaga sinensis COⅠspecific loci. The reaction system containing template, primers, Bst DNA polymerase and methyl red-phenol red indicator amplified at 63 ℃ for 90 min. The LAMP results were determined by visual observation and compared with DNA barcoding and polymerase chain reaction (PCR) methods to investigate the specificity and sensitivity. Results: All 11 batches of the authentic Eupolyphaga sinensis sample tubes colour changed from purple red to orange yellow, while sample tubes of 1 batch of Steleophaga plancyi, 9 batches Opisthoplatia orientalis and 1 batch of the adulterant, Cybister tripunctatus orientalis, remained purple red after the reaction. The LAMP results were consistent with those of DNA barcoding and PCR. The detection limit of LAMP was 0.984 ng·mL-1. Conclusion: The established LAMP method is accurate, sensitive and easy to operate, low equipment required, and can be applied to the rapid screening of the authentication of Eupolyphaga sinensis.

, correspAuthors=Su-mei LI, 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=Wei-tao CHEN, Yang-xue LI, Jie-yi JIANG, Can-hui XIE, De-zheng JIA, Xiao-tong LIU, Hui-na LIAO, Su-mei LI), CN=ArticleExt(id=1240688797638258758, articleId=1240688794454782890, tenantId=1146029695717560320, journalId=1205117023404326918, language=CN, title=地鳖的环介导等温扩增快速鉴别, columnId=1206272758971953622, journalTitle=药物分析杂志, columnName=标准研讨, runingTitle=null, highlight=null, articleAbstract=

目的：建立基于环介导等温扩增技术（LAMP）的地鳖分子鉴定方法。方法：针对地鳖COⅠ序列设计特异性引物2对（外引物DB-F3、DB-B3和内引物DB-FIP、DB-BIP），在含有Bst DNA聚合酶和甲基红-苯酚红指示剂的反应体系下于63 ℃下恒温扩增90 min，通过目测检视判断反应结果，与DNA条形码和聚合酶链式反应（PCR）鉴定方法进行对比，考察方法专属性和灵敏度。结果：11批地鳖样品均由紫红色变为橙黄色，1批冀地鳖、9批金边土鳖和1批伪品龙虱均为紫红色，鉴别结果与DNA条形码，PCR结果一致，LAMP检出限为0.984 ng·mL-1。结论：本项目建立的LAMP检测方法准确，灵敏度高，操作简便快捷，对设备要求低，可应用于地鳖鉴别的快速筛查。

, correspAuthors=李素梅, authorNote=null, correspAuthorsNote=
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J Fujian Norm Univ (Nat Sci Ed)，2024，40(1)：34, articleTitle=RPA-based rapid diagnostic strategies for pathogens, refAbstract=null)], funds=[Fund(id=1240704458158567654, tenantId=1146029695717560320, journalId=1205117023404326918, articleId=1240688794454782890, awardId=20244001, language=CN, fundingSource=广东省中医药局科研项目(20244001), fundOrder=null, country=null), Fund(id=1240704458267619565, tenantId=1146029695717560320, journalId=1205117023404326918, articleId=1240688794454782890, awardId=粤财科教[2021]113号, language=CN, fundingSource=2021年省属科研机构稳定性支持项目(粤财科教[2021]113号), fundOrder=null, country=null)], companyList=[AuthorCompany(id=1240704449354724235, tenantId=1146029695717560320, journalId=1205117023404326918, articleId=1240688794454782890, xref=1., ext=[AuthorCompanyExt(id=1240704449363112845, tenantId=1146029695717560320, journalId=1205117023404326918, articleId=1240688794454782890, companyId=1240704449354724235, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=1.Guangdong Second Traditional Chinese Medicine Hospital (Guangdong Province Engineering Technology Research Institute of Traditional Chinese Medicine), Guangdong Provincial Key Laboratory of Research and Development in Traditional Chinese Medicine, Guangzhou, Guangdong 510095, China), AuthorCompanyExt(id=1240704449371501455, tenantId=1146029695717560320, journalId=1205117023404326918, articleId=1240688794454782890, companyId=1240704449354724235, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=1.广东省第二中医院（广东省中医药工程技术研究院）　广东省中医药研究开发重点实验室，广州　510095)]), AuthorCompany(id=1240704449463776149, tenantId=1146029695717560320, journalId=1205117023404326918, articleId=1240688794454782890, xref=2., ext=[AuthorCompanyExt(id=1240704449472164757, tenantId=1146029695717560320, journalId=1205117023404326918, articleId=1240688794454782890, companyId=1240704449463776149, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=2.The School of the Fifth Clinical Medicine, Guangzhou University of Chinese Medicine, Guangzhou 510405, China), AuthorCompanyExt(id=1240704449476359062, tenantId=1146029695717560320, journalId=1205117023404326918, articleId=1240688794454782890, companyId=1240704449463776149, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=2.广州中医药大学　第五临床医学院，广州　510405)]), AuthorCompany(id=1240704449572828060, tenantId=1146029695717560320, journalId=1205117023404326918, articleId=1240688794454782890, xref=3., ext=[AuthorCompanyExt(id=1240704449581216670, tenantId=1146029695717560320, journalId=1205117023404326918, articleId=1240688794454782890, companyId=1240704449572828060, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=3.School of Pharmacy, Guangzhou Xinhua University, Guangzhou 510520, China), AuthorCompanyExt(id=1240704449589605279, tenantId=1146029695717560320, journalId=1205117023404326918, articleId=1240688794454782890, companyId=1240704449572828060, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=3.广州新华学院　药学院，广州　510520)])], figs=[ArticleFig(id=1240704455310635162, tenantId=1146029695717560320, journalId=1205117023404326918, articleId=1240688794454782890, language=EN, label=Fig.1, caption=Phylogenetic tree of Eupolyphaga sinensis and the adulterants, figureFileSmall=gBEuRM4dFHZjEExGZIiEgQ==, figureFileBig=EaWGoHyzP9n0gUgGRpsL3w==, tableContent=null), ArticleFig(id=1240704455390326941, tenantId=1146029695717560320, journalId=1205117023404326918, articleId=1240688794454782890, language=CN, label=图1, caption=地鳖和混伪品系统发育树, figureFileSmall=gBEuRM4dFHZjEExGZIiEgQ==, figureFileBig=EaWGoHyzP9n0gUgGRpsL3w==, tableContent=null), ArticleFig(id=1240704455516156067, tenantId=1146029695717560320, journalId=1205117023404326918, articleId=1240688794454782890, language=EN, label=Fig.2, caption=PCR result of Eupolyphaga sinensis and the adulterants, figureFileSmall=V6HJvXINRmKj8XB/eLllaw==, figureFileBig=r/1fL+zBNhemwbXcaE+YUA==, tableContent=null), ArticleFig(id=1240704455595847848, tenantId=1146029695717560320, journalId=1205117023404326918, articleId=1240688794454782890, language=CN, label=图2, caption=地鳖和混伪品PCR结果

M.DNA标尺（DNA ladder）

, figureFileSmall=V6HJvXINRmKj8XB/eLllaw==, figureFileBig=r/1fL+zBNhemwbXcaE+YUA==, tableContent=null), ArticleFig(id=1240704455679733933, tenantId=1146029695717560320, journalId=1205117023404326918, articleId=1240688794454782890, language=EN, label=Fig.3, caption=Specific loci of Eupolyphaga sinensis and the LAMP primers position, figureFileSmall=Q+ClRW2VOTybk6sjOxJvxA==, figureFileBig=VVOaaGIImMHqVzBp3Sx5JQ==, tableContent=null), ArticleFig(id=1240704455767814321, tenantId=1146029695717560320, journalId=1205117023404326918, articleId=1240688794454782890, language=CN, label=图3, caption=地鳖特异性位点和LAMP引物位置, figureFileSmall=Q+ClRW2VOTybk6sjOxJvxA==, figureFileBig=VVOaaGIImMHqVzBp3Sx5JQ==, tableContent=null), ArticleFig(id=1240704455851700404, tenantId=1146029695717560320, journalId=1205117023404326918, articleId=1240688794454782890, language=EN, label=Fig.4, caption=LAMP reaction result of Eupolyphaga sinensis and the adulterants, figureFileSmall=sDJvrj/EIEjD82FXh2BwlQ==, figureFileBig=EvuUfY3UwdvbnMsgRcEdkw==, tableContent=null), ArticleFig(id=1240704455910420665, tenantId=1146029695717560320, journalId=1205117023404326918, articleId=1240688794454782890, language=CN, label=图4, caption=地鳖和混伪品LAMP反应结果

1~11.地鳖（Eupolyphaga sinensis）　12.冀地鳖[Steleophaga plancyi (Boleny)]　13~21.金边土鳖（Opisthoplatia orientalis）　22.东方潜龙虱（Cybister tripunctatus orientalis）　B.空白对照（black control）

, figureFileSmall=sDJvrj/EIEjD82FXh2BwlQ==, figureFileBig=EvuUfY3UwdvbnMsgRcEdkw==, tableContent=null), ArticleFig(id=1240704455998501054, tenantId=1146029695717560320, journalId=1205117023404326918, articleId=1240688794454782890, language=EN, label=Fig.5, caption=Sensitivity of Eupolyphaga sinensis LAMP, figureFileSmall=LiYUswhsQBpq8l0jBSuaEQ==, figureFileBig=d991hlfFQ397bNTHu1PCCw==, tableContent=null), ArticleFig(id=1240704456099164356, tenantId=1146029695717560320, journalId=1205117023404326918, articleId=1240688794454782890, language=CN, label=图5, caption=地鳖LAMP灵敏度检测结果

A. 984 0 ng·mL-1　B. 984 ng·mL-1　C. 98.4 ng·mL-1　D. 9.84 ng·mL-1　E. 0.984ng·mL-1　F. 0.098 4 ng·mL-1　Blk.空白对照（black control）

, figureFileSmall=LiYUswhsQBpq8l0jBSuaEQ==, figureFileBig=d991hlfFQ397bNTHu1PCCw==, tableContent=null), ArticleFig(id=1240704456208216263, tenantId=1146029695717560320, journalId=1205117023404326918, articleId=1240688794454782890, language=EN, label=Tab.1, caption=

Primers of Eupolyphaga sinensis DNA barcoding identification and LAMP

, figureFileSmall=null, figureFileBig=null, tableContent=
引物
（primer）		序列（5’→3’）
[sequence (5’→3’)]
HCO2198TAAACTTCAGGGTGACCAAAAAATCA
LCO1490GGTCAACAAATCATAAAGATATTGG
DB-F3CCTGATATAGCTTTTCCTCGT
DB-B3CCTAAAATTGATCTAACCCCC
DB-FIPGCCTCTTTCAACTATACTGCTTGTTTTTGATTATTACCACCATCCC
DB-BIPCACAGGTTGGACTGTTTACCCACGTAAGGAAAAAATTGCTAAATCG
), ArticleFig(id=1240704456296296653, tenantId=1146029695717560320, journalId=1205117023404326918, articleId=1240688794454782890, language=CN, label=表1, caption=

地鳖DNA条形码鉴别和LAMP反应引物

, figureFileSmall=null, figureFileBig=null, tableContent=
引物
（primer）		序列（5’→3’）
[sequence (5’→3’)]
HCO2198TAAACTTCAGGGTGACCAAAAAATCA
LCO1490GGTCAACAAATCATAAAGATATTGG
DB-F3CCTGATATAGCTTTTCCTCGT
DB-B3CCTAAAATTGATCTAACCCCC
DB-FIPGCCTCTTTCAACTATACTGCTTGTTTTTGATTATTACCACCATCCC
DB-BIPCACAGGTTGGACTGTTTACCCACGTAAGGAAAAAATTGCTAAATCG
), ArticleFig(id=1240704457701388495, tenantId=1146029695717560320, journalId=1205117023404326918, articleId=1240688794454782890, language=EN, label=Tab.2, caption=

Eupolyphaga sinensis samples information

, figureFileSmall=null, figureFileBig=null, tableContent=
编号(No.)来源(source)产地(habitat)编号(No.)来源(source)产地(habitat)
1地鳖(Eupolyphaga sinensis)河北(Hebei)12冀地鳖[Steleophaga plancyi (Boleny)]山西(Shangxi)
2地鳖(Eupolyphaga sinensis)山东(Shandong)13金边土鳖(Opisthoplatia orientalis)广东(Guangdong)
3地鳖(Eupolyphaga sinensis)山东(Shandong)14金边土鳖(Opisthoplatia orientalis)广西(Guangxi)
4地鳖(Eupolyphaga sinensis)河北(Hebei)15金边土鳖(Opisthoplatia orientalis)广西(Guangxi)
5地鳖(Eupolyphaga sinensis)河北(Hebei)16金边土鳖(Opisthoplatia orientalis)广西(Guangxi)
6地鳖(Eupolyphaga sinensis)江苏(Hebei)17金边土鳖(Opisthoplatia orientalis)浙江(Zhejiang)
7地鳖(Eupolyphaga sinensis)山东(Shandong)18金边土鳖(Opisthoplatia orientalis)湖北(Hubei)
8地鳖(Eupolyphaga sinensis)湖南(Hunan)19金边土鳖(Opisthoplatia orientalis)广西(Guangxi)
9地鳖(Eupolyphaga sinensis)河南(Henan)20金边土鳖(Opisthoplatia orientalis)江西(Jiangxi)
10地鳖(Eupolyphaga sinensis)河南(Henan)21金边土鳖(Opisthoplatia orientalis)浙江(Zhejiang)
11地鳖(Eupolyphaga sinensis)江苏(Jiangsu)22东方潜龙虱(Cybister tripunctatus orientalis)安徽(Anhui)
), ArticleFig(id=1240704457802051798, tenantId=1146029695717560320, journalId=1205117023404326918, articleId=1240688794454782890, language=CN, label=表2, caption=

地鳖样品信息

, figureFileSmall=null, figureFileBig=null, tableContent=
编号(No.)来源(source)产地(habitat)编号(No.)来源(source)产地(habitat)
1地鳖(Eupolyphaga sinensis)河北(Hebei)12冀地鳖[Steleophaga plancyi (Boleny)]山西(Shangxi)
2地鳖(Eupolyphaga sinensis)山东(Shandong)13金边土鳖(Opisthoplatia orientalis)广东(Guangdong)
3地鳖(Eupolyphaga sinensis)山东(Shandong)14金边土鳖(Opisthoplatia orientalis)广西(Guangxi)
4地鳖(Eupolyphaga sinensis)河北(Hebei)15金边土鳖(Opisthoplatia orientalis)广西(Guangxi)
5地鳖(Eupolyphaga sinensis)河北(Hebei)16金边土鳖(Opisthoplatia orientalis)广西(Guangxi)
6地鳖(Eupolyphaga sinensis)江苏(Hebei)17金边土鳖(Opisthoplatia orientalis)浙江(Zhejiang)
7地鳖(Eupolyphaga sinensis)山东(Shandong)18金边土鳖(Opisthoplatia orientalis)湖北(Hubei)
8地鳖(Eupolyphaga sinensis)湖南(Hunan)19金边土鳖(Opisthoplatia orientalis)广西(Guangxi)
9地鳖(Eupolyphaga sinensis)河南(Henan)20金边土鳖(Opisthoplatia orientalis)江西(Jiangxi)
10地鳖(Eupolyphaga sinensis)河南(Henan)21金边土鳖(Opisthoplatia orientalis)浙江(Zhejiang)
11地鳖(Eupolyphaga sinensis)江苏(Jiangsu)22东方潜龙虱(Cybister tripunctatus orientalis)安徽(Anhui)
), ArticleFig(id=1240704457894326492, tenantId=1146029695717560320, journalId=1205117023404326918, articleId=1240688794454782890, language=EN, label=Tab.3, caption=

Genbank BLAST result of Eupolyphaga sinensis samples

, figureFileSmall=null, figureFileBig=null, tableContent=
编号
(No.)		匹配核酸片段登录号
( accession)		核酸片段来源物种
(organism)		得分
(score)		E值
(E value)		序列鉴别率
(per. ident)/%
1KJ616712.1地鳖(Eupolyphaga sinensis)1 1750100
2KJ616712.1地鳖(Eupolyphaga sinensis)1 1880100
3KJ616712.1地鳖(Eupolyphaga sinensis)1 1570100
4KJ616712.1地鳖(Eupolyphaga sinensis)1 1570100
5JF700163.1地鳖(Eupolyphaga sinensis)1 131099.52
6KJ616712.1地鳖(Eupolyphaga sinensis)1 164099.84
7KJ616712.1地鳖(Eupolyphaga sinensis)1 1860100
8KJ616712.1地鳖(Eupolyphaga sinensis)1 1700100
9KJ616712.1地鳖(Eupolyphaga sinensis)1 1490100
10KJ616712.1地鳖(Eupolyphaga sinensis)1 1530100
11KJ616712.1地鳖(Eupolyphaga sinensis)1 1510100
12NC_049567.1冀地鳖(Polyphaga plancyi)1 194099.85
13NC_029225.1金边土鳖(Opisthoplatia orientalis)1 1600100
14NC_029225.1金边土鳖(Opisthoplatia orientalis)1 1680100
15NC_029225.1金边土鳖(Opisthoplatia orientalis)1 1570100
16NC_029225.1金边土鳖(Opisthoplatia orientalis)1 1420100
17NC_029225.1金边土鳖(Opisthoplatia orientalis)1 1640100
18NC_029225.1金边土鳖(Opisthoplatia orientalis)1 1810100
19NC_029225.1金边土鳖(Opisthoplatia orientalis)1 1620100
20NC_029225.1金边土鳖(Opisthoplatia orientalis)1 149099.68
21NC_029225.1金边土鳖(Opisthoplatia orientalis)1 1570100
22GU681182.1善泅龙虱(Dytiscus dauricus)811091.07
), ArticleFig(id=1240704457986601181, tenantId=1146029695717560320, journalId=1205117023404326918, articleId=1240688794454782890, language=CN, label=表3, caption=

地鳖样品Genbank数据库比对结果

, figureFileSmall=null, figureFileBig=null, tableContent=
编号
(No.)		匹配核酸片段登录号
( accession)		核酸片段来源物种
(organism)		得分
(score)		E值
(E value)		序列鉴别率
(per. ident)/%
1KJ616712.1地鳖(Eupolyphaga sinensis)1 1750100
2KJ616712.1地鳖(Eupolyphaga sinensis)1 1880100
3KJ616712.1地鳖(Eupolyphaga sinensis)1 1570100
4KJ616712.1地鳖(Eupolyphaga sinensis)1 1570100
5JF700163.1地鳖(Eupolyphaga sinensis)1 131099.52
6KJ616712.1地鳖(Eupolyphaga sinensis)1 164099.84
7KJ616712.1地鳖(Eupolyphaga sinensis)1 1860100
8KJ616712.1地鳖(Eupolyphaga sinensis)1 1700100
9KJ616712.1地鳖(Eupolyphaga sinensis)1 1490100
10KJ616712.1地鳖(Eupolyphaga sinensis)1 1530100
11KJ616712.1地鳖(Eupolyphaga sinensis)1 1510100
12NC_049567.1冀地鳖(Polyphaga plancyi)1 194099.85
13NC_029225.1金边土鳖(Opisthoplatia orientalis)1 1600100
14NC_029225.1金边土鳖(Opisthoplatia orientalis)1 1680100
15NC_029225.1金边土鳖(Opisthoplatia orientalis)1 1570100
16NC_029225.1金边土鳖(Opisthoplatia orientalis)1 1420100
17NC_029225.1金边土鳖(Opisthoplatia orientalis)1 1640100
18NC_029225.1金边土鳖(Opisthoplatia orientalis)1 1810100
19NC_029225.1金边土鳖(Opisthoplatia orientalis)1 1620100
20NC_029225.1金边土鳖(Opisthoplatia orientalis)1 149099.68
21NC_029225.1金边土鳖(Opisthoplatia orientalis)1 1570100
22GU681182.1善泅龙虱(Dytiscus dauricus)811091.07
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地鳖的环介导等温扩增快速鉴别
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陈伟韬 1 , 李养学 1 , 江洁怡 1 , 谢灿辉 2 , 贾德政 2 , 刘晓彤 3 , 廖惠娜 3 , 李素梅 1, **
药物分析杂志 | 标准研讨 2024,44(9): 1529-1534
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药物分析杂志 | 标准研讨 2024, 44(9): 1529-1534
地鳖的环介导等温扩增快速鉴别
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陈伟韬1 , 李养学1, 江洁怡1, 谢灿辉2, 贾德政2, 刘晓彤3, 廖惠娜3, 李素梅1, **
作者信息
  • 1.广东省第二中医院（广东省中医药工程技术研究院）　广东省中医药研究开发重点实验室，广州　510095
  • 2.广州中医药大学　第五临床医学院，广州　510405
  • 3.广州新华学院　药学院，广州　510520

    • Tel: (020)83482098; E-mail:

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** Tel: (020)83482098; E-mail:
Rapid authentication of Eupolyphaga sinensis by loop-mediated isothermal amplification
Wei-tao CHEN1 , Yang-xue LI1, Jie-yi JIANG1, Can-hui XIE2, De-zheng JIA2, Xiao-tong LIU3, Hui-na LIAO3, Su-mei LI1, **
Affiliations
  • 1.Guangdong Second Traditional Chinese Medicine Hospital (Guangdong Province Engineering Technology Research Institute of Traditional Chinese Medicine), Guangdong Provincial Key Laboratory of Research and Development in Traditional Chinese Medicine, Guangzhou, Guangdong 510095, China
  • 2.The School of the Fifth Clinical Medicine, Guangzhou University of Chinese Medicine, Guangzhou 510405, China
  • 3.School of Pharmacy, Guangzhou Xinhua University, Guangzhou 510520, China
出版时间: 2024-09-30 doi: 10.16155/j.0254-1793.2024-0035
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摘要
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目的：建立基于环介导等温扩增技术（LAMP）的地鳖分子鉴定方法。方法：针对地鳖COⅠ序列设计特异性引物2对（外引物DB-F3、DB-B3和内引物DB-FIP、DB-BIP），在含有Bst DNA聚合酶和甲基红-苯酚红指示剂的反应体系下于63 ℃下恒温扩增90 min，通过目测检视判断反应结果，与DNA条形码和聚合酶链式反应（PCR）鉴定方法进行对比，考察方法专属性和灵敏度。结果：11批地鳖样品均由紫红色变为橙黄色，1批冀地鳖、9批金边土鳖和1批伪品龙虱均为紫红色，鉴别结果与DNA条形码，PCR结果一致，LAMP检出限为0.984 ng·mL-1。结论：本项目建立的LAMP检测方法准确，灵敏度高，操作简便快捷，对设备要求低，可应用于地鳖鉴别的快速筛查。

地鳖  /  环介导等温扩增技术  /  DNA条形码  /  聚合酶链式反应  /  中药材鉴别  /  目视检查  /  快速鉴别

Objective: To establish an authentication method for Eupolyphaga sinensis based on loop-mediated isothermal amplification (LAMP). Methods: Two pairs of primers （outer primer DB-F3, DB-B3 and inner primer DB-FIP, DB-BIP） were designed according to Eupolyphaga sinensis COⅠspecific loci. The reaction system containing template, primers, Bst DNA polymerase and methyl red-phenol red indicator amplified at 63 ℃ for 90 min. The LAMP results were determined by visual observation and compared with DNA barcoding and polymerase chain reaction (PCR) methods to investigate the specificity and sensitivity. Results: All 11 batches of the authentic Eupolyphaga sinensis sample tubes colour changed from purple red to orange yellow, while sample tubes of 1 batch of Steleophaga plancyi, 9 batches Opisthoplatia orientalis and 1 batch of the adulterant, Cybister tripunctatus orientalis, remained purple red after the reaction. The LAMP results were consistent with those of DNA barcoding and PCR. The detection limit of LAMP was 0.984 ng·mL-1. Conclusion: The established LAMP method is accurate, sensitive and easy to operate, low equipment required, and can be applied to the rapid screening of the authentication of Eupolyphaga sinensis.

Eupolyphaga sinensis  /  loop-mediated isothermal amplification  /  DNA barcoding  /  polymerase chain reaction  /  authentication of Chinese medicinal materials  /  visual check  /  rapid identification
陈伟韬, 李养学, 江洁怡, 谢灿辉, 贾德政, 刘晓彤, 廖惠娜, 李素梅. 地鳖的环介导等温扩增快速鉴别. 药物分析杂志, 2024 , 44 (9) : 1529 -1534 . DOI: 10.16155/j.0254-1793.2024-0035
Wei-tao CHEN, Yang-xue LI, Jie-yi JIANG, Can-hui XIE, De-zheng JIA, Xiao-tong LIU, Hui-na LIAO, Su-mei LI. Rapid authentication of Eupolyphaga sinensis by loop-mediated isothermal amplification[J]. Chinese Journal of Pharmaceutical Analysis, 2024 , 44 (9) : 1529 -1534 . DOI: 10.16155/j.0254-1793.2024-0035
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中药土鳖虫古称“䗪虫”，药用历史超过2000年，具有活血散瘀、消癥破坚、疗伤定痛的功效，在治疗血瘀经闭、癥瘕积聚、跌打损伤、瘀血凝痛方面疗效良好[1-3]。土鳖虫入药来源有地鳖Eupolyphaga sinensis Walker和冀地鳖Steleophaga plancyi (Boleny) [Polyphaga plancyi Bolivar]，岭南地区有以金边土鳖Opisthoplatia orientalis Burm.作土鳖虫使用，此外在江苏部分地区有写“䗪虫”但实为东方潜龙虱Cybister tripunctatus orientalis Gschwendtn，为土鳖虫伪品[4-6]。金边土鳖和地鳖、冀地鳖作用相近，但临床功效存在一定差异，龙虱性味功效完全不同，不得混用，故建立该品种鉴别方法对于规范用药，保障临床疗效具有积极意义。现有药材质量标准难以区分土鳖虫及其混伪品，通过DNA条形码技术成功区分地鳖和金边土鳖，但该法检测时间长，需DNA条形码数据库和测序平台支持，不利于推广应用[7]。环介导等温扩增技术（LAMP）是通过对6个特定的序列区域设计2对特异性引物，在Bst DNA聚合酶的作用下可恒温进行的基因扩增技术[8-11]，已在姜黄、白花蛇舌草、藏红花、蕲蛇、地龙等多种中药材鉴别研究中应用[12-21]。本研究针对地鳖与其他来源土鳖虫、伪品在COⅠ序列的特异性位点设计引物，进行LAMP反应，通过甲基红-苯酚红指示剂显色检视，建立地鳖LAMP快速鉴别方法。
1　仪器与试药
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1.1　仪器
数字加热摇动金属浴（Thermo Scientific公司）；96孔热循环仪（Applied Biosystems公司）；电泳仪（Bio-Rad公司）；Qubit 3.0荧光分光光度计（Life Technologies公司）；TLC Visualizer薄层色谱数码成像系统（CAMAG公司）。
1.2　试药
DP304 DNA提取试剂盒（北京天根生化科技有限公司）；Colorimetric LAMP RY试剂盒（珠海宝锐生物科技有限公司）；Premix Taq预混型Ex Taq酶（Takara公司）；gelred核酸染料（Biotium公司）；引物由深圳华大基因科技有限公司合成，引物序列见表1。
药材样品经广东省中医药工程技术研究院副主任中药师李素梅鉴定，各药材样品包含地鳖Eupolyphaga sinensis Walker的雌虫干燥体，冀地鳖Steleophaga plancyi (Boleny)的雌虫干燥体，金边土鳖Opisthoplatia orientalis Burm.的干燥体和东方潜龙虱Cybister tripunctatus orientalis Gschwendtn的干燥体，样品信息详见表2。
2　方法与结果
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2.1　DNA提取
各药材样品用研钵碾碎，按DP304 DNA提取试剂盒说明书步骤提取样品DNA。
2.2　基于COⅠ的DNA条形码鉴别
参考2020年版《中华人民共和国药典》中药材DNA条形码分子鉴定法指导原则，以COⅠ序列引物HCO2198、LCO1490对各样品DNA进行PCR扩增，PCR产物双向测序，拼接的序列通过BLAST在线比对GenBank数据库。结果11批地鳖、1批冀地鳖、9批金边土鳖样品DNA条形码均与数据库中匹配的核酸片段物种相对应，但龙虱样品匹配的核酸片段非对应物种，序列鉴别率较低，见表3。运用MEGA 7软件以邻接法基于K2P模型构建系统发育树（Bootstrap检测数1 000）分析，土鳖虫正品和混伪品在根部已分为不同分支，可作区分，各样品依物种形成分支，与样品信息对应，其中地鳖与冀地鳖亲缘关系较近，见图1。
2.3　PCR鉴别
PCR反应于200 μL离心管进行，体系总体积50 μL，包括Premix Taq预混型Ex Taq酶（含Mg2+ 4 mmol·L-1，dNTPs 400 μmol·L-1）25 μL，引物DB-F3、DB-B3（10 μmol·L-1）各1 μL，模版5 μL，无菌超纯水18 μL，阴性对照以无菌超纯水作为模版。PCR扩增条件：94 ℃预变性30 s，98 ℃变性10 s、60 ℃退火30 s、72 ℃延伸60 s循环反应30次，最后72 ℃延伸5 min。电泳检测条件：2%琼脂糖凝胶加入gelred核酸染料，样品PCR溶液上样量10 μL，100 V电泳40 min，于TLC Visualizer薄层色谱数码成像系统检视。结果地鳖在200 bp左右有单一条带，冀地鳖、金边土鳖和龙虱对应位置无条带，见图2。
2.3　LAMP鉴别
2.3.1　LAMP引物设计
以地鳖DNA条形码序列为模版，通过在线工具PrimerExplorer V5（http：//primerexplorer.jp/lampv5e/index.html）对F3、F2、F1c、B3、B2、B1c等6个序列设计引物，根据地鳖、冀地鳖与金边土鳖、龙虱基因的差异位点，对设计的引物进行筛选、优化，得到引物DB-F3、DB-B3、DB-FIP、DB-BIP，见图3。
2.3.2　LAMP反应
LAMP反应于200 μL离心管进行，体系总体积25 μL，包括Colorimetric LAMP RY试剂盒中的2×LAMP Premix Buffer（含甲酚红-苯酚红指示剂）12.5 μL和Bst 2.0 HS DNA聚合酶（8 U·μL-1）1 μL，LAMP引物F3、B3（10 μmol·L-1）各0.5 μL和FIP、BIP（10 μmol·L-1）各4 μL，模版2 μL，无菌超纯水0.5 μL，阴性对照以无菌超纯水作为模版。反应离心管置金属浴，63 ℃恒温扩增90 min，检视离心管颜色。对收集的多批地鳖及混伪品样品进行检测，结果11批地鳖样品溶液颜色由紫红色变为橙黄色，1批冀地鳖样品、9批金边土鳖样品、1批龙虱样品和阴性对照为紫红色，成功将地鳖正品与其他样品区分，结果与DNA条形码鉴别一致，见图4。
3　讨论
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DNA条形码技术鉴别样品，可给出物种来源，但必须依赖可靠的样品DNA序列和数据库，故龙虱未能成功匹配可能与提取的基因质量、COⅠ序列关于该物种的鉴别适用性以及数据库所收录核酸片段的代表性和数量有关。PCR鉴别在初期试验有部分批次地鳖未能扩增出条带，冀地鳖出现非特异性扩增，可能是模版量和PCR扩增条件不合适导致，优化相关条件后，所有地鳖样品均扩增出条带，能与其他来源土鳖虫及伪品进行区分。
根据上述LAMP检测结果，该法专属性良好，与DNA条形码和PCR方法相当。取第11批样品DNA以Qubit荧光计测定浓度为9 840 ng·mL-1，以无菌超纯水稀释成984、98.4、9.84、0.984、0.098 4 ng·mL-1等不同浓度，按“2.3.2”项方法进行反应，检测灵敏度，结果浓度稀释至0.984 ng·mL-1时仍明显变色，见图5。Bst DNA聚合酶在60~65 ℃酶活性良好，本研究分别比较了在60、63、65 ℃下扩增情况，结果扩增90 min后，地鳖样品均变为黄色，说明在60~65 ℃温度变化对扩增影响不大。研究还考察了不同扩增时间，结果地鳖样品在约40至50 min开始变色，到80至90 min变为橙黄色至黄色，与混伪品颜色区分明显，故本鉴别方法扩增时间确定为90 min。
LAMP反应体系通过Bst DNA聚合酶能在恒温条件下高效扩增，因此可使用金属浴代替较昂贵的PCR仪实现反应，同时DNA模版量需求少。DNA复制产生焦磷酸根[22]，使得反应管内pH下降，在甲基红-苯酚红指示剂作用下产生由紫红色逐渐变为黄色的颜色变化，因此发生扩增的样品可通过肉眼检视结果，无需进行电泳检测及使用凝胶成像系统、荧光检测仪、浊度分析仪等设备。
本研究建立了基于LAMP的地鳖分子生物学快速鉴定方法，该法专属性强，耐用性较好，具有便捷、高效、仪器设备要求低的优点，为开发地鳖现场快速筛查方法提供一种新途径。
基金
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  • 广东省中医药局科研项目(20244001)
  • 2021年省属科研机构稳定性支持项目(粤财科教[2021]113号)
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2024年第44卷第9期
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doi: 10.16155/j.0254-1793.2024-0035
  • 接收时间：2024-01-18
  • 首发时间：2026-03-17
  • 出版时间：2024-09-30
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  • 收稿日期：2024-01-18
基金
广东省中医药局科研项目(20244001)
2021年省属科研机构稳定性支持项目(粤财科教[2021]113号)
作者信息
    1.广东省第二中医院（广东省中医药工程技术研究院）　广东省中医药研究开发重点实验室，广州　510095
    2.广州中医药大学　第五临床医学院，广州　510405
    3.广州新华学院　药学院，广州　510520

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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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