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Article(id=1217529307346420254, tenantId=1146029695717560320, journalId=1149652044408987649, issueId=1217529305693864468, articleNumber=null, orderNo=null, doi=10.19812/j.cnki.jfsq11-5956/ts.20250415003, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=research-article, receivedDate=1744646400000, receivedDateStr=2025-04-15, revisedDate=null, revisedDateStr=null, acceptedDate=null, acceptedDateStr=null, onlineDate=1768211207472, onlineDateStr=2026-01-12, pubDate=1752508800000, pubDateStr=2025-07-15, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1768211207472, onlineIssueDateStr=2026-01-12, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1768211207472, creator=13701087609, updateTime=1768211207472, updator=13701087609, issue=Issue{id=1217529305693864468, tenantId=1146029695717560320, journalId=1149652044408987649, year='2025', volume='16', issue='13', pageStart='1', pageEnd='320', issueExtLink='null', onlineDate='null', pubDate='null', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=null, createTime=1768211207077, creator=13701087609, updateTime=1768212057891, updator=13701087609, preIssue=null, nextIssue=null, ext={EN=IssueExt(id=1217532874337730593, tenantId=1146029695717560320, journalId=1149652044408987649, issueId=1217529305693864468, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1217532874337730594, tenantId=1146029695717560320, journalId=1149652044408987649, issueId=1217529305693864468, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null}, startPage=18, endPage=25, ext={EN=ArticleExt(id=1217529307623244329, articleId=1217529307346420254, tenantId=1146029695717560320, journalId=1149652044408987649, language=EN, title=Detection of Oncorhynchus mykiss source components based on improved pre-amplification real-time quantitative polymerase chain reaction method, columnId=1217529307430306336, journalTitle=Journal of Food Safety & Quality, columnName=Highlight: Processing and Quality Safety of Aquatic Products, runingTitle=null, highlight=null, articleAbstract=

Objective To establish a pre-amplified quantitative real-time polymerase chain reaction (qPCR) method for the detection of Oncorhynchus mykiss source components. Methods The study developed an enhanced pre-amplified qPCR method, which was based on the validation and improvement of the existing standard qPCR method and compared the performance of both methods. Results Specificity testing of the standard qPCR method revealed cross-reactivity with closely related species, such as Coho salmon and Atlantic salmon. The established improved pre-amplified qPCR method, which incorporated a pre-amplification step, exhibited high specificity and no cross amplification was observed for Coho salmon, Atlantic salmon and other closely related species. The amplification efficiencies of the standard qPCR and the pre-amplified qPCR methods were 90.25% and 104.91%, respectively. The lowest limits of detection of the 2 kinds of methods were 2.99×10-3 ng/μL (0.83-8.87 pg/μL, 95% confidence interval) for the standard qPCR and 1.50×10-4 ng/μL (0.09-0.27 pg/μL, 95% confidence interval) for the pre-amplified qPCR. Conclusion The method established in this study has the characteristics of strong specificity, high sensitivity, and is suitable for authenticity and adulteration detection of Oncorhynchus mykiss source components in food products.

, correspAuthors=Yan-Hong LIU, Qiu-Yue 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=Duo-Lan BAI, Lin HONG, Di YANG, Wen-Chao CHEN, Jia-Qi WANG, Lei FANG, Yan-Hong LIU, Qiu-Yue ZHENG), CN=ArticleExt(id=1217529310638948996, articleId=1217529307346420254, tenantId=1146029695717560320, journalId=1149652044408987649, language=CN, title=基于改进的预扩增实时荧光定量聚合酶链式反应法检验虹鳟源性成分, columnId=1217529307631632938, journalTitle=食品安全质量检测学报, columnName=本期重点：水产品加工与质量安全, runingTitle=null, highlight=null, articleAbstract=

目的 建立改进的预扩增实时荧光定量聚合酶链式反应法(quantitative real-time polymerase chain reaction, qPCR)法检验虹鳟源性成分的方法。方法 基于对现行标准中虹鳟qPCR方法进行验证和改进提高, 建立改进的预扩增qPCR方法, 并对两种qPCR方法进行比较。结果 经验证, 在采用标准中qPCR方法对虹鳟的近种鱼种银鲑、大西洋鲑等鱼种进行特异性验证时有交叉反应, 基于设置预扩增反应步骤的改进qPCR方法特异性良好, 对于银鲑、大西洋鲑等近种鱼种未出现交叉扩增。qPCR和预扩增qPCR方法的扩增效率分别为90.25%、104.91%, 最低检出限分别为2.99×10-3 ng/μL (0.83~8.87 pg/μL, 95%置信区间)和1.50×10-4 ng/μL (0.09~0.27 pg/μL, 95%置信区间)。结论 所改进的虹鳟源性成分预扩增qPCR方法具有高特异性、高灵敏度, 适用于食品样品中虹鳟源性成分的真实性和掺假检验。

, correspAuthors=刘彦泓, 郑秋月, authorNote=null, correspAuthorsNote=
*刘彦泓(1976—), 女, 硕士, 正高级工程师, 主要研究方向为食品安全检测。E-mail: ;
郑秋月(1972—), 女, 博士, 研究员, 主要研究方向为食品质量与安全。E-mail:
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白朵兰(2000—), 女, 硕士研究生, 主要研究方向为食品质量与安全。E-mail:

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白朵兰(2000—), 女, 硕士研究生, 主要研究方向为食品质量与安全。E-mail:

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白朵兰(2000—), 女, 硕士研究生, 主要研究方向为食品质量与安全。E-mail:

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Foods, 2021, 10(11): 2631-2631., articleTitle=A multiplex PCR assay combined with capillary electrophoresis for the simultaneous identification of atlantic cod, pacific cod, blue whiting, haddock, and alaska pollock, refAbstract=null), Reference(id=1217901269553959112, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1217529307346420254, doi=null, pmid=null, pmcid=null, year=2020, volume=101, issue=5, pageStart=1792, pageEnd=1799, url=null, language=null, rfNumber=[29], rfOrder=33, authorNames=YAO L, XIN HM, QU M, journalName=Journal of the Science of Food and Agriculture, refType=null, unstructuredReference=YAO L, XIN HM, QU M, et al. Development of duplex Real-timepPolymerase chain reaction for simultaneous detection of oilfish and escolar derived components[J]. Journal of the Science of Food and Agriculture, 2020, 101(5): 1792-1799., articleTitle=Development of duplex Real-timepPolymerase chain reaction for simultaneous detection of oilfish and escolar derived components, refAbstract=null), Reference(id=1217901269646233804, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1217529307346420254, doi=null, pmid=null, pmcid=null, year=2007, volume=7, issue=4, pageStart=544, pageEnd=548, url=null, language=null, rfNumber=[30], rfOrder=34, authorNames=IVANOVA VN, ZEMLAK ST, HANNER H, journalName=Molecular Ecology Notes, refType=null, unstructuredReference=IVANOVA VN, ZEMLAK ST, HANNER H, et al. Universal primer cocktails for fish DNA barcoding[J]. Molecular Ecology Notes, 2007, 7(4): 544-548., articleTitle=Universal primer cocktails for fish DNA barcoding, refAbstract=null)], funds=[Fund(id=1217901264625651749, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1217529307346420254, awardId=2023MK130, language=CN, fundingSource=国家市场监管总局科技计划项目(2023MK130), fundOrder=null, country=null)], companyList=[AuthorCompany(id=1217901254576099868, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1217529307346420254, xref=1, ext=[AuthorCompanyExt(id=1217901254580294172, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1217529307346420254, companyId=1217901254576099868, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=1 Key Laboratory of Biotechnology and Bioresources Utilization of Ministry of Education, College of Life Science, Dalian Minzu University, Dalian 116600, China), AuthorCompanyExt(id=1217901254588682781, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1217529307346420254, companyId=1217901254576099868, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=1 大连民族大学生命科学学院, 生物技术与资源利用教育部重点实验室, 大连 116600)]), AuthorCompany(id=1217901254697734694, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1217529307346420254, xref=2, ext=[AuthorCompanyExt(id=1217901254710317608, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1217529307346420254, companyId=1217901254697734694, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=2 Dalian Center for Certification and Food and Drug Control, Technology Innovation Center of Food Safety Technique of Inspection for State Market Regulation (Rapid Screening and Traceability for Edible Agricultural Product Safety), Dalian 116630, China), AuthorCompanyExt(id=1217901254718706217, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1217529307346420254, companyId=1217901254697734694, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=2 大连市检验检测认证技术服务中心, 国家市场监督管理总局技术创新中心(食用农产品安全快速检测与追溯), 大连 116630)])], figs=[ArticleFig(id=1217901260955636638, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1217529307346420254, language=EN, label=Fig.1, caption=Sequence alignment results of primers and probe for Oncorhynchus mykiss source components in standard method, figureFileSmall=xovVn7p2vE96Vl+DFLzmNQ==, figureFileBig=iBA6GGyFeFcFxJ9aF8pJEA==, tableContent=null), ArticleFig(id=1217901261094048680, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1217529307346420254, language=CN, label=图1, caption=标准方法中虹鳟源性成分引物和探针序列比对结果, figureFileSmall=xovVn7p2vE96Vl+DFLzmNQ==, figureFileBig=iBA6GGyFeFcFxJ9aF8pJEA==, tableContent=null), ArticleFig(id=1217901261270209463, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1217529307346420254, language=EN, label=Fig.2, caption=Specific detection of Oncorhynchus mykiss source components of universal qPCR (A) and pre-amplified qPCR (B), figureFileSmall=/1jWlsXauVCJhElDlonI/A==, figureFileBig=36oKrmUXGZFtD2OolfhYew==, tableContent=null), ArticleFig(id=1217901261387649981, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1217529307346420254, language=CN, label=图2, caption=qPCR (A)和预扩增qPCR (B)检验虹鳟源性成分特异性

注: A. 标准中的qPCR反应程序, 在第30个循环开始, 银鲑、大西洋鲑、红鲑、粉鲑、秋鲑等样品基因组DNA均出现非特异性荧光扩增曲线; B. 预扩增qPCR反应程序, 仅虹鳟基因组DNA出现特异性扩增, 其余样品基因组DNA均无非特异性扩增。

, figureFileSmall=/1jWlsXauVCJhElDlonI/A==, figureFileBig=36oKrmUXGZFtD2OolfhYew==, tableContent=null), ArticleFig(id=1217901261484118980, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1217529307346420254, language=EN, label=Fig.3, caption=Sensitivity results of Oncorhynchus mykiss source components of universal qPCR (A) and pre-amplified qPCR (B), figureFileSmall=Dw0XucgNT+oasktT5vaYjg==, figureFileBig=rcXfIeviMUeBz5wFyWm6AA==, tableContent=null), ArticleFig(id=1217901262822101964, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1217529307346420254, language=CN, label=图3, caption=qPCR (A)和预扩增qPCR (B)检验虹鳟源性成分扩增效率分析, figureFileSmall=Dw0XucgNT+oasktT5vaYjg==, figureFileBig=rcXfIeviMUeBz5wFyWm6AA==, tableContent=null), ArticleFig(id=1217901262960514005, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1217529307346420254, language=EN, label=Fig.4, caption=Sensitivity of qPCR and pre-amplification qPCR in detecting genomic DNA of Oncorhynchus mykiss, figureFileSmall=5v+tCVBmWh0DGd+ClABviQ==, figureFileBig=Nw6E7U869XruM9yf1mUCwQ==, tableContent=null), ArticleFig(id=1217901263094731741, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1217529307346420254, language=CN, label=图4, caption=qPCR和预扩增qPCR检验虹鳟基因组DNA的灵敏度, figureFileSmall=5v+tCVBmWh0DGd+ClABviQ==, figureFileBig=Nw6E7U869XruM9yf1mUCwQ==, tableContent=null), ArticleFig(id=1217901263199589345, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1217529307346420254, language=EN, label=Fig.5, caption=Determination of LOD values for qPCR (A) and pre-amplified qPCR (B), figureFileSmall=6ZjMAcReEyPHK7k5NtOBIQ==, figureFileBig=ZscfUo8g4PnPVo4zS1s8iQ==, tableContent=null), ArticleFig(id=1217901263333807077, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1217529307346420254, language=CN, label=图5, caption=qPCR (A)和预扩增qPCR (B)的LOD值确定, figureFileSmall=6ZjMAcReEyPHK7k5NtOBIQ==, figureFileBig=ZscfUo8g4PnPVo4zS1s8iQ==, tableContent=null), ArticleFig(id=1217901263488996335, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1217529307346420254, language=EN, label=Table 1, caption=

Gene barcode sequencing primer sequence and PCR reaction conditions

, figureFileSmall=null, figureFileBig=null, tableContent=
基因 引物名称 引物序列(5′→3′) 反应条件 条带大小/bp
COI 上游引物F TTCTCCACCAACCACAARGAYATYGG 94 ℃ 30 s; 94 ℃ 10 s, 60 ℃ 30 s, 72 ℃ 45 s, 40个循环; 72 ℃ 10 min 655
下游引物R CACCTCAGGGTGTCCGAARAAYCARAA
), ArticleFig(id=1217901263623214070, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1217529307346420254, language=CN, label=表1, caption=

基因条形码测序引物序列及PCR反应条件

, figureFileSmall=null, figureFileBig=null, tableContent=
基因 引物名称 引物序列(5′→3′) 反应条件 条带大小/bp
COI 上游引物F TTCTCCACCAACCACAARGAYATYGG 94 ℃ 30 s; 94 ℃ 10 s, 60 ℃ 30 s, 72 ℃ 45 s, 40个循环; 72 ℃ 10 min 655
下游引物R CACCTCAGGGTGTCCGAARAAYCARAA
), ArticleFig(id=1217901263765820411, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1217529307346420254, language=EN, label=Table 2, caption=

Primers/probes used in the qPCR method

, figureFileSmall=null, figureFileBig=null, tableContent=
引物/探针 引物/探针序列(5'→3') 参考文献
Few AGTAATTATCGGCATACGAAACCAG [28]
Rev GTTTCGATAATGATCAGTACTGGGATC
Probe FAM-CTACGGCCGCCCTCGGCCATTTAT-TAMRA
), ArticleFig(id=1217901263879066622, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1217529307346420254, language=CN, label=表2, caption=

用于qPCR方法的引物/探针

, figureFileSmall=null, figureFileBig=null, tableContent=
引物/探针 引物/探针序列(5'→3') 参考文献
Few AGTAATTATCGGCATACGAAACCAG [28]
Rev GTTTCGATAATGATCAGTACTGGGATC
Probe FAM-CTACGGCCGCCCTCGGCCATTTAT-TAMRA
), ArticleFig(id=1217901264009089031, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1217529307346420254, language=EN, label=Table 3, caption=

Sequencing results of sample DNA gene barcode

, figureFileSmall=null, figureFileBig=null, tableContent=
编号 样品名称 拉丁文名称 测序结果 测序结果是否与样品名称一致
(genbank序列号)
1 大西洋鲑 Salmo salar KX145289.1 是
2 银鲑 Oncorhynchus kisutch FJ998805.1 是
3 虹鳟 Oncorhynchus mykiss EU752133.1 是
4 粉鲑 Oncorhynchus gorbuscha KX145377.1 是
5 红鲑 Oncorhynchus nerka FJ999169.1 是
6 秋鲑 Oncorhynchus keta AP010773.1 是
), ArticleFig(id=1217901264118140942, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1217529307346420254, language=CN, label=表3, caption=

样品DNA基因条形码测序结果

, figureFileSmall=null, figureFileBig=null, tableContent=
编号 样品名称 拉丁文名称 测序结果 测序结果是否与样品名称一致
(genbank序列号)
1 大西洋鲑 Salmo salar KX145289.1 是
2 银鲑 Oncorhynchus kisutch FJ998805.1 是
3 虹鳟 Oncorhynchus mykiss EU752133.1 是
4 粉鲑 Oncorhynchus gorbuscha KX145377.1 是
5 红鲑 Oncorhynchus nerka FJ999169.1 是
6 秋鲑 Oncorhynchus keta AP010773.1 是
), ArticleFig(id=1217901264248164371, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1217529307346420254, language=EN, label=Table 4, caption=

Reaction program conditions of universal qPCR and pre-amplified qPCR

, figureFileSmall=null, figureFileBig=null, tableContent=
检验方法 温度/℃ 时间 循环数/个
qPCR 95 10 min 1
95 15 s 40
60 1 min
预扩增qPCR 95 10 s 15
50 10 s
95 1 min 1
95 10 s 40
55 30 s
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qPCR和预扩增qPCR反应程序

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检验方法 温度/℃ 时间 循环数/个
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95 15 s 40
60 1 min
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50 10 s
95 1 min 1
95 10 s 40
55 30 s
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基于改进的预扩增实时荧光定量聚合酶链式反应法检验虹鳟源性成分
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白朵兰 1 , 洪麟 2 , 杨滴 2 , 陈文超 2 , 王佳琪 2 , 方蕾 1 , 刘彦泓 2, * , 郑秋月 1, *
食品安全质量检测学报 | 本期重点：水产品加工与质量安全 2025,16(13): 18-25
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食品安全质量检测学报 | 本期重点：水产品加工与质量安全 2025, 16(13): 18-25
基于改进的预扩增实时荧光定量聚合酶链式反应法检验虹鳟源性成分
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白朵兰1 , 洪麟2, 杨滴2, 陈文超2, 王佳琪2, 方蕾1, 刘彦泓2, * , 郑秋月1, *
作者信息
  • 1 大连民族大学生命科学学院, 生物技术与资源利用教育部重点实验室, 大连 116600
  • 2 大连市检验检测认证技术服务中心, 国家市场监督管理总局技术创新中心(食用农产品安全快速检测与追溯), 大连 116630

    • 白朵兰(2000—), 女, 硕士研究生, 主要研究方向为食品质量与安全。E-mail:

通讯作者:

*刘彦泓(1976—), 女, 硕士, 正高级工程师, 主要研究方向为食品安全检测。E-mail: ;
郑秋月(1972—), 女, 博士, 研究员, 主要研究方向为食品质量与安全。E-mail:
Detection of Oncorhynchus mykiss source components based on improved pre-amplification real-time quantitative polymerase chain reaction method
Duo-Lan BAI1 , Lin HONG2, Di YANG2, Wen-Chao CHEN2, Jia-Qi WANG2, Lei FANG1, Yan-Hong LIU2, * , Qiu-Yue ZHENG1, *
Affiliations
  • 1 Key Laboratory of Biotechnology and Bioresources Utilization of Ministry of Education, College of Life Science, Dalian Minzu University, Dalian 116600, China
  • 2 Dalian Center for Certification and Food and Drug Control, Technology Innovation Center of Food Safety Technique of Inspection for State Market Regulation (Rapid Screening and Traceability for Edible Agricultural Product Safety), Dalian 116630, China
出版时间: 2025-07-15 doi: 10.19812/j.cnki.jfsq11-5956/ts.20250415003
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摘要
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目的 建立改进的预扩增实时荧光定量聚合酶链式反应法(quantitative real-time polymerase chain reaction, qPCR)法检验虹鳟源性成分的方法。方法 基于对现行标准中虹鳟qPCR方法进行验证和改进提高, 建立改进的预扩增qPCR方法, 并对两种qPCR方法进行比较。结果 经验证, 在采用标准中qPCR方法对虹鳟的近种鱼种银鲑、大西洋鲑等鱼种进行特异性验证时有交叉反应, 基于设置预扩增反应步骤的改进qPCR方法特异性良好, 对于银鲑、大西洋鲑等近种鱼种未出现交叉扩增。qPCR和预扩增qPCR方法的扩增效率分别为90.25%、104.91%, 最低检出限分别为2.99×10-3 ng/μL (0.83~8.87 pg/μL, 95%置信区间)和1.50×10-4 ng/μL (0.09~0.27 pg/μL, 95%置信区间)。结论 所改进的虹鳟源性成分预扩增qPCR方法具有高特异性、高灵敏度, 适用于食品样品中虹鳟源性成分的真实性和掺假检验。

虹鳟源性成分  /  真实性检验  /  实时荧光定量聚合酶链式反应法  /  预扩增实时荧光定量聚合酶链式反应法

Objective To establish a pre-amplified quantitative real-time polymerase chain reaction (qPCR) method for the detection of Oncorhynchus mykiss source components. Methods The study developed an enhanced pre-amplified qPCR method, which was based on the validation and improvement of the existing standard qPCR method and compared the performance of both methods. Results Specificity testing of the standard qPCR method revealed cross-reactivity with closely related species, such as Coho salmon and Atlantic salmon. The established improved pre-amplified qPCR method, which incorporated a pre-amplification step, exhibited high specificity and no cross amplification was observed for Coho salmon, Atlantic salmon and other closely related species. The amplification efficiencies of the standard qPCR and the pre-amplified qPCR methods were 90.25% and 104.91%, respectively. The lowest limits of detection of the 2 kinds of methods were 2.99×10-3 ng/μL (0.83-8.87 pg/μL, 95% confidence interval) for the standard qPCR and 1.50×10-4 ng/μL (0.09-0.27 pg/μL, 95% confidence interval) for the pre-amplified qPCR. Conclusion The method established in this study has the characteristics of strong specificity, high sensitivity, and is suitable for authenticity and adulteration detection of Oncorhynchus mykiss source components in food products.

Oncorhynchus mykiss source  /  authenticity identification  /  quantitative real-time polymerase chain reaction  /  pre-amplified quantitative real-time polymerase chain reaction
白朵兰, 洪麟, 杨滴, 陈文超, 王佳琪, 方蕾, 刘彦泓, 郑秋月. 基于改进的预扩增实时荧光定量聚合酶链式反应法检验虹鳟源性成分. 食品安全质量检测学报, 2025 , 16 (13) : 18 -25 . DOI: 10.19812/j.cnki.jfsq11-5956/ts.20250415003
Duo-Lan BAI, Lin HONG, Di YANG, Wen-Chao CHEN, Jia-Qi WANG, Lei FANG, Yan-Hong LIU, Qiu-Yue ZHENG. Detection of Oncorhynchus mykiss source components based on improved pre-amplification real-time quantitative polymerase chain reaction method[J]. Journal of Food Safety & Quality, 2025 , 16 (13) : 18 -25 . DOI: 10.19812/j.cnki.jfsq11-5956/ts.20250415003
正文
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0 引言
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三文鱼是鲑科鱼类的统称, 包括大西洋鲑、虹鳟、银鲑、王鲑、红鲑、秋鲑、粉鲑等, 最早在分类学上是指鲑科鱼鲑属(Salmo)的大西洋鲑(Salmo salar)[1]。三文鱼因口感独特、营养丰富, 含有大量不饱和脂肪酸和人体必需微量元素[2], 具有高经济价值。
虹鳟(Oncorhynchus mykiss), 俗名淡水三文鱼, 属鲑形目, 鲑亚科, 大麻哈鱼属(Oncorhynchus)[3]。虹鳟鱼虽在肉质、纹理、口感和大西洋鲑极其相似, 但在营养价值和经济价值上与大西洋鲑鱼存在较大差异[4]。据报道, 在淡水中养殖的虹鳟鱼存在感染寄生虫[1]和过量砷积累[5]的风险, 生食但不标识容易产生食品安全隐患。在高额利润的驱动下, 一些不法商贩常以低价虹鳟鱼替代大西洋鲑鱼进行售卖, 导致三文鱼近种鱼间的掺假现象在生产加工及商品销售环节屡见不鲜。这些欺诈事件损害消费者经济利益, 同时加大食品安全风险。丁清龙等[1]采用实时荧光聚合酶链式反应(polymerase chain reaction, PCR)技术检测广东省内销售的三文鱼产品, 从标识含有大西洋鲑的98批次样品中检出了虹鳟源性成分。CAWTHORN等[6]报道了发生在南非的因鱼类命名错误或标签错误造成的海鲜欺诈案例。HORREO等[7]调查西班牙马德里餐馆, 发现秋鲑掺假冒充大西洋鲑鱼。美国华盛顿地区38%的三文鱼水产品存在商品标识与实际品种不符的问题[8]。为了保护消费者权益以及确保公众健康, 迫切需要一种精准可靠的方法进行三文鱼品种真实性检验。
在水产养殖、原料采购、生产加工环节, 虹鳟鱼和大西洋鲑鱼等其他三文鱼的鉴定主要依赖于其形态特征, 而经加工制作成寿司和鱼片等产品后, 很难根据残余特征鉴别三文鱼原料真伪[9]。国家食品安全标准规定预包装产品的标签应符合GB 7718—2011《食品安全国家标准 预包装食品标签通则》的要求, 标注原料鱼产地以及种名, 标注三文鱼(大西洋鲑)、三文鱼(虹鳟)等。目前鱼类种类鉴定方法主要有酶联免疫吸附法和蛋白质组学法[10-11]、分子生物学方法[12-14]、代谢组学技术[15]等。CHAI等[16]使用UV-Vis光谱仪鉴别鱼种类。CHEN等[17]建立大西洋鲑鱼拉曼光谱掺假鉴别方法。MA等[18]开发了数字PCR定量检测虹鳟鱼和大西洋鲑鱼的方法。YANG等[19]建立了大西洋鲑鱼和虹鳟鱼重组酶聚合酶扩增结合侧向流试纸条(recombinase polymerase amplification combined with lateral flow strip, RPA-LFS)鉴定方法。基于核酸的分子生物学方法广泛应用于鱼类真实性及掺假检验[20-22]。其中实时荧光定量PCR (real-time fluorescent quantitative PCR, qPCR)是物种真实性鉴别的金标准, 常被制定为检测标准[23]。qPCR应用于物种鉴定更加稳定, 方法特异性和灵敏度都比较高[24]。
基因条形码技术是鱼种鉴定常用方法[25-29], 本研究先采用基因条形码利用一段标准DNA序列[30]进行物种鉴定鉴别实验用鱼类样品的物种真实性和准确性。现行检验虹鳟源性成分的标准方法为SN/T 5480—2022《虹鳟成分鉴定技术规范实时荧光PCR法》, 本研究在应用该标准检验食品中虹鳟源性成分时发现, 标准中规定的qPCR方法存在非特异性扩增。为了提高虹鳟源性成分检测的准确性, 本研究改进标准中的qPCR反应程序, 设置预扩增反应步骤, 用以加速样本核酸裂解和富集模板, 建立虹鳟源性成分的预扩增qPCR方法, 消除了现行标准方法中存在的非特异性扩增的问题, 并对低浓度样品的做到了高灵敏特异鉴定, 实现食品中虹鳟源性成分的快速可靠检验。
1 材料与方法
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1.1 材料、试剂与仪器
1.1.1 实验样品
用于方法建立及特异性实验用的虹鳟、银鲑、大西洋鲑、红鲑、粉鲑、秋鲑等样品, 来源于大连市水产品生产加工企业, 样品的物种真实性通过DNA条形码进行测序确认。同时收集了37份标识含有虹鳟、大西洋鳟、银鲑、粉鲑、红鲑、秋鲑等成分的食品样本, 来源于大连市水产品生产加工企业及各大超市出售的产品。
1.1.2 试剂与仪器
基因组DNA提取试剂[货号: AS1600, 普洛麦格(北京)生物技术有限公司]; qPCR反应试剂[货号: Code No. RR390A, 宝生物工程(大连)有限公司]; QRT-PCR实时荧光PCR试剂(货号: Cat# 6001001101, 卡尤迪生物科技有限公司); 测序试剂盒(BigDye Terminators V3.0, 上海嘉鹏科技有限公司)。
NanoDrop超微量分光光度计、ABI QuantStudio 7定量PCR 仪、LLSC009高速冷冻离心机、ABI3130X基因测序分析仪(美国赛默飞世尔科技公司); BWS-10恒温水槽与水浴锅(上海一恒科学仪器有限公司); PTX-FA210S分析天平(精度0.1 mg, 福建华志电子科技有限公司); Synergy H1 酶标仪(美国伯腾仪器有限公司)。
1.2 方法
1.2.1 样品处理及基因组DNA提取
提取虹鳟及其他近种鱼的基因组DNA用于qPCR分析实验。取200 mg组织肉, 采用磁珠法DNA提取试剂盒提取鱼类基因组DNA。采用超微量分光光度计根据A260/A280比值测定并计算提取的基因组DNA的纯度和浓度。
1.2.2 样品真实属性基因条形码测试
根据国际生物条形码协会推荐的鱼类DNA条形码基因并参考相关文献资料, 基于鱼类COI基因序列测序结果对样品进行物种鉴定, 保证实验样品的真实属性。采用鱼类DNA条形码通用引物扩增三文鱼线粒体COI基因序列[30]。DNA条形码通用引物信息如表1所示。
按照表1中反应条件进行基因条形码PCR扩增, PCR产物用2%琼脂糖凝胶电泳回收纯化, 使用ABI测序仪进行双向测序, 使用SeqMan 11.1对序列峰图进行校对拼接, 分析测序结果。
1.2.3 qPCR引物与探针
qPCR扩增的引物和Taq Man探针(Fw/Rev/probe)基于已发布实施的SN/T 5480—2022, 使用primer blast (https://blast.ncbi.nlm.nih.gov/Blast.cgi)进行评估。引物和探针均由宝生物工程(大连)有限公司合成, 具体序列见表2。
1.3 qPCR 方法验证及改进
1.3.1 标准方法qPCR扩增
基于标准方法中的qPCR反应体积25 μL, 其中12.5 μL含有酶混合物的Probe qPCR Mix, 0.4 μmol/L正向引物、反向引物、探针各1 μL, 7.5 μL无DNA酶的超纯水, 2 μL提取的DNA模板。qPCR反应程序: 95 ℃ 10 min, 1个循环; 95 ℃ 15 s, 60 ℃ 1 min, 40个循环; 在60 ℃时收集FAM荧光信号, 反应结束后分析荧光扩增曲线结果。
1.3.2 预扩增qPCR扩增
预扩增qPCR反应体系为25 μL, 其中16 μL含有酶混合物的Probe qPCR Mix, 0.4 μmol/L正向引物(Fw)、反向引物(Rev)、探针(Probe)各1 μL, 4 μL无DNA酶的超纯水, 2 μL提取的DNA模板。改进的预扩增qPCR反应程序: 首先进行预扩增步骤, 95 ℃ 10 s, 50 ℃ 10 s, 15个循环, 此步骤不采集FAM荧光信号; 95 ℃ 1 min; 95 ℃ 10 s, 55 ℃ 30 s, 40个循环, 在55 ℃时采集FAM荧光信号, 反应结束后分析荧光扩增曲线结果。
1.3.3 qPCR和预扩增qPCR特异性分析
使用标准中虹鳟源性成分的引物和探针(表2), 针对虹鳟同属或近属的其他鱼种的基因组DNA进行交叉反应分析, 包括银鲑、大西洋鲑、红鲑、粉鲑、秋鲑基因组DNA, 用来评估qPCR和预扩增qPCR的特异性。超纯水作为空白对照。
1.3.4 qPCR和预扩增qPCR的扩增效率分析
以提取的虹鳟基因组DNA(初始质量浓度为2.99×101 ng/μL)为模板, 进行10倍浓度稀释, 分别制备了2.99×100、2.99×10-1、2.99×10-2、2.99×10-3 、2.99×10-4 ng/μL总计共6个质量浓度梯度稀释液, 测试qPCR和预扩增qPCR方法的扩增效率。每个质量浓度设3个平行反应, 分别采用2种方法重复测试3次。以起始样品DNA质量浓度的稀释倍数为横坐标(X, ng/μL), 以扩增的Ct值为纵坐标(Y), 分别绘制线性曲线, 计算分析得到相应的线性方程及其斜率、线性相关系数(r2)和PCR反应效率(E), 探讨两种qPCR扩增方法的扩增效率。用公式E=10(-1/斜率)-1计算qPCR效率(E), 并以百分比表示。E是评价PCR检验性能最重要的指标之一, 需要满足达到90%~110%的要求。
1.3.5 qPCR和预扩增qPCR灵敏度分析
为了进一步确定qPCR和预扩增qPCR的最低检出限(limit of detection, LOD), 将提取的虹鳟基因组DNA稀释为2.99×10-2、2.99×10-3、1.50×10-3、1.50×10-4、7.48×10-5、5.98×10-5、2.99×10-5 ng/μL共计7个质量浓度梯度, 每个质量浓度梯度平行测试12次, 以12个子样全部为阳性结果的浓度确定LOD。
1.3.6 基于预扩增qPCR方法检验实际样本
本研究收集了37份标识含有虹鳟、大西洋鲑、银鲑、粉鲑、王鲑等成分的食品样本, 评估改进的预扩增qPCR方法在实际样品检验中的适用性, 每个样本进行3次重复试验。所收集样品涵盖常见的水产品及其加工产品类别, 包括原料、半加工产品、深加工食品等。
1.4 数据处理
为了测定分析虹鳟源性成分的灵敏度, LOD数据以Ct值的平均值±标准偏差表示, 使用GraphPad prism 9.3进行半对数回归分析评估。使用MedCalc 20.0软件对连续稀释7个质量浓度梯度的重复测试数据进行概率回归分析, 计算qPCR和预扩增qPCR在95%概率水平下的LOD。LOD是评价方法有效检验范围和准确定量范围的重要参数, 分析过程中在满足95%置信区间的条件下所能检验到的目标样品的最低浓度。
2 结果与分析
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2.1 样品真实性基因条形码测试结果
利用基因条形码技术对收集到的三文鱼样品的COI基因部分特异区段进行测序, 测序结果在美国国家生物技术信息中心(National Center for Biotechnology Information,NCBI) (http://www.ncbi.nlm.nih.gov)进行blast比对, 得到样品基因条形码的测序结果为: 大西洋鲑(KX145289.1)、银鲑(FJ998805.1)、虹鳟(EU752133.1)、粉鲑(KX145377.1)、红鲑(FJ999169.1)、秋鲑(AP010773.1), 基因序列相似性均高达99%, 结果表明样品来源真实准确。所收集的虹鳟、银鲑、大西洋鲑、红鲑、粉鲑、秋鲑等样品基因条形码测试结果如表3所示。
2.2 引物和探针序列特异性比对
以虹鳟的线粒体基因片段(genbank: LC050735.1)为靶标基因片段, 对标准中的qPCR引物(Fw/Rev)和Taq Man探针(probe)在NCBI进行同源性比对, 比对结果表明该引物和探针对于虹鳟源性成分的检验具有较好的特异性(图1)。基于应用标准方法检测三文鱼真实属性时发现存在非特异性扩增, 本研究进一步采用实际样品验证标准方法的特异性。
2.3 qPCR扩增特异性结果
使用标准中的引物和探针及反应程序(qPCR)进行特异性实验, 发现虹鳟的近属三文鱼源性成分样品, 包括银鲑、大西洋鲑、红鲑、粉鲑及秋鲑的基因组DNA均发生非特异性扩增, 在第30个PCR循环后均出现阳性荧光扩增曲线(图2A)。为了消除非特异性扩增, 本研究对标准中的qPCR方法进行改进, 增设了一个15个循环的预扩增PCR反应过程。优化后的qPCR热循环条件为先进行预扩增95 ℃ 10, 50 ℃ 10 s, 15个循环, 不采集FAM荧光信号; 然后再进行实时荧光PCR扩增, 95 ℃ 1 min; 95 ℃ 10 s, 55 ℃ 30 s, 40个循环, 在55 ℃时采集FAM荧光信号(表4)。
2.4 预扩增qPCR特异性结果
采用标准中的qPCR引物探针, 基于本研究改进的预扩增qPCR进行虹鳟成分的特异性试验。预扩增qPCR结果如图2B所示。结果表明仅虹鳟基因组DNA出现特异性荧光扩增曲线, 银鲑、大西洋鲑、红鲑、粉鲑、秋鲑等鱼种基因组DNA均未出现特异性扩增, 没有发生交叉反应。表明改进的预扩增qPCR反应程序具有良好的特异性。
2.5 qPCR和预扩增qPCR的扩增效率结果
以梯度稀释的虹鳟基因组DNA(初始质量浓度为2.99×101 ng/μL)为模板, 评估qPCR以及改进的预扩增qPCR方法扩增效率。分别以模板DNA浓度的稀释度为横坐标(X, ng/μL), 以扩增的Ct值为纵坐标(Y), 使用GraphPad prism 9.3软件绘制线性曲线。使用Origin 2021绘制qPCR的线性曲线, 得到Y=-3.58X+24.12, 相关系数r2=0.99, 由扩增效率公式[E=10(-1/斜率)-1]计算得到E=90.25%(图3A)。绘制预扩增qPCR的线性曲线, 得到Y=-3.21X+10.01, 相关系数r2=0.99, 计算得到E=104.91% (图3B)。
2.6 qPCR和预扩增qPCR的灵敏度结果
对提取的虹鳟基因组DNA进行灵敏度实验。qPCR可以检验到虹鳟基因组DNA的质量浓度为2.99×10-3 ng/μL, 3个平行样品中仅有2个阳性, 预扩增qPCR可以检验到虹鳟基因组DNA的质量浓度为1.50×10-4 ng/μL, 3个平行样品都为阳性结果(图4)。
并用MedCalc 22.0软件对结果进行概率回归分析, 以计算qPCR和预扩增qPCR在95%概率水平下的检验低限。在95%概率水平下, qPCR检验虹鳟基因组DNA的LOD为2.99×10-3 ng/μL, 95%的置信区间为0.83~8.87 pg/μL(图5A)。预扩增qPCR检验虹鳟基因组DNA的LOD为1.50×10-4 ng/μL, 95%的置信区间为0.09~0.27 pg/μL(图5B)。
2.7 预扩增qPCR检验实际样品结果
为了评价本研究改进的预扩增qPCR检验实际样品中虹鳟源性成分的有效性, 本研究检验了37份标识含有虹鳟、大西洋鲑、银鲑、粉鲑、王鲑等成分的水产类食品样品提取的基因组DNA。使用预扩增qPCR方法, 在标识含有虹鳟源性成分的19份样本中均检出阳性扩增, Ct值为15.02~25.46, 与样品标识相符; 其中有2份鱼丸样品没有标识含有虹鳟源性成分, 仍然检出虹鳟源性成分阳性, 与样品标识不符。所有检出虹鳟源性成分的样品均采用基因条形码进行测序进行阳性检出验证, 测序结果均与预扩增qPCR检验结果一致。另外16份样本未检出虹鳟源性成分, 没有出现Ct数值, 检验结果为阴性, 与样品标识相符。此外, 本实验室留存的已经过形态学鉴别和基因条形码测序验证为含有虹鳟源性成分的5份样品, 经预扩增qPCR方法检测均出现虹鳟源性成分的特异性扩增阳性结果, 进一步验证了方法的可靠性与适用性。
3 结论与讨论
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本研究开发了一种改进提高的预扩增qPCR方法, 通过在实时荧光PCR扩增反应之前增加15个循环预扩增来加速模板的热裂解, 加速模板DNA热变性和复性, 此阶段不收集荧光信号, 使核酸充分释放出来, 富集模板DNA, 累积了更多的模板用于后续的qPCR分析, 有利于低拷贝检测的高阈值判读, 在qPCR扩增阶段早期观察到阳性荧光信号, 提高特异性扩增效率。克服了SN/T 5480—2022 qPCR方法检测大西洋鲑、银鲑、红鲑、粉鲑、秋鲑等源性成分发生非特异性扩增的问题, 有效解决了由于三文鱼鱼种之间基因组序列极为相似, 易出现PCR扩增交叉反应, 影响物种真实属性鉴定准确性的难题。并且改进的预扩增qPCR方法最低LOD为1.50×10-4 ng/μL (0.09~0.27 pg/μL, 95%置信区间), 扩增效率为104.91%; 优于标准方法qPCR的2.99×10-3 ng/μL灵敏度(0.83~8.87 pg/μL), 扩增效率90.25%。同时进行了37份水产品样品的预扩增qPCR分析, 包括原料、单一含量食品、混合食品、以及深加工食品等不同种类样品。本研究分别采用预扩增qPCR方法检测实际样品和已知样品, 对于出现虹鳟源性成分阳性扩增的样品同时进行基因条形码测序, 验证方法的准确性。基于两种形式的验证结果表明本研究建立的基于改进的预扩增qPCR方法稳定可靠, 适用于食品样品中虹鳟源性成分的真实性和掺假检验。
基金
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  • 国家市场监管总局科技计划项目(2023MK130)
文献
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2025年第16卷第13期
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文章信息
doi: 10.19812/j.cnki.jfsq11-5956/ts.20250415003
  • 接收时间：2025-04-15
  • 首发时间：2026-01-12
  • 出版时间：2025-07-15
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  • 收稿日期：2025-04-15
基金
国家市场监管总局科技计划项目(2023MK130)
作者信息
    1 大连民族大学生命科学学院, 生物技术与资源利用教育部重点实验室, 大连 116600
    2 大连市检验检测认证技术服务中心, 国家市场监督管理总局技术创新中心(食用农产品安全快速检测与追溯), 大连 116630

通讯作者:

*刘彦泓(1976—), 女, 硕士, 正高级工程师, 主要研究方向为食品安全检测。E-mail: ;
郑秋月(1972—), 女, 博士, 研究员, 主要研究方向为食品质量与安全。E-mail:
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https://castjournals.cast.org.cn/joweb/spaq/CN/10.19812/j.cnki.jfsq11-5956/ts.20250415003
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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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