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Article(id=1153433739548942903, tenantId=1146029695717560320, journalId=1149652044408987649, issueId=1153433737141412332, articleNumber=null, orderNo=null, doi=10.19812/j.cnki.jfsq11-5956/ts.20241130002, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=null, receivedDate=1732896000000, receivedDateStr=2024-11-30, revisedDate=null, revisedDateStr=null, acceptedDate=null, acceptedDateStr=null, onlineDate=1752929633271, onlineDateStr=2025-07-19, pubDate=1745510400000, pubDateStr=2025-04-25, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1752929633271, onlineIssueDateStr=2025-07-19, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1752929633271, creator=13701087609, updateTime=1752929633271, updator=13701087609, issue=Issue{id=1153433737141412332, tenantId=1146029695717560320, journalId=1149652044408987649, year='2025', volume='16', issue='8', pageStart='1', pageEnd='316', issueExtLink='null', onlineDate='null', pubDate='null', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=0, createTime=1752929632696, creator=13701087609, updateTime=1757293087150, updator=13701087609, preIssue=null, nextIssue=null, ext={EN=IssueExt(id=1171735391666225233, tenantId=1146029695717560320, journalId=1149652044408987649, issueId=1153433737141412332, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1171735391666225234, tenantId=1146029695717560320, journalId=1149652044408987649, issueId=1153433737141412332, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null}, startPage=261, endPage=266, ext={EN=ArticleExt(id=1153433740073230923, articleId=1153433739548942903, tenantId=1146029695717560320, journalId=1149652044408987649, language=EN, title=Application of colloidal gold immunochromatography for detecting various animal derived components in meat, columnId=1151895321388347923, journalTitle=Journal of Food Safety & Quality, columnName=Food Analysis and Detection, runingTitle=null, highlight=null, articleAbstract=

Objective To study the accuracy and feasibility of colloidal gold immunochromatography for the detection of animal-derived components in meat products. Methods The rapid detection kit was used to detect the animal-derived components in meat products. The sensitivity, specificity, adulteration simulation, false positive rate and false negative rate of 8 kinds of animal-derived components were tested, and quantitative real-time polymerase chain reaction (QPCR) was used as a reference. Results The limits of detection of the rapid detection kit could reach 0.1% for cattle, chicken, duck and goose, and 0.01% for pig, sheep, horse and donkey. The limits of detection of pig, cattle, sheep, chicken, duck, goose, horse and donkey in the adulterated simulation could reach 0.5%, and there was no cross-reaction with other non-species channel detection, and the specificity was good. Conclusion The colloidal gold immunochromatography method established in this study is suitable for the rapid screening and detection of animal origin in meat products.

, correspAuthors=Jun LUAN, 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=Yu-Rong JIANG, Li MA, Wei ZHANG, Qing-Fei LI, Jun LUAN), CN=ArticleExt(id=1153433746045920067, articleId=1153433739548942903, tenantId=1146029695717560320, journalId=1149652044408987649, language=CN, title=应用胶体金免疫层析法检测肉类中多种动物源性成分, columnId=1151895321958773274, journalTitle=食品安全质量检测学报, columnName=食品分析与检测, runingTitle=null, highlight=null, articleAbstract=

目的 研究胶体金免疫层析法检测肉制品中动物源性成分的准确性及可行性。方法 利用该快速检测试剂盒检测肉制品中动物源性成分, 对样品中8种动物源成分的灵敏度、特异性、掺假模拟、假阳性率、假阴性率进行相关实验, 并以实时荧光定量聚合酶链式反应(quantitative real-time polymerase chain reaction, QPCR)为参比对照。结果 该快速检测试剂盒的灵敏度对牛、鸡、鸭、鹅的检出限可达到0.1%, 对猪、羊、马、驴的检出限可达到0.01%; 掺假模拟中猪、牛、羊、鸡、鸭、鹅、马、驴的检出限均可达到0.5%, 同时与其他非本物种通道检测时均无交叉反应, 特异性较好。结论 本研究建立的胶体金免疫层析法适用于肉制品中动物源性的现场快速筛查和检测。

, correspAuthors=栾军, authorNote=null, correspAuthorsNote=
* 栾军(1982—), 男, 硕士, 工程师, 主要研究方向为食品安全检测。E-mail:
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姜玉榕(1997—), 女, 主要研究方向为食品安全检测。E-mail:

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姜玉榕(1997—), 女, 主要研究方向为食品安全检测。E-mail:

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姜玉榕(1997—), 女, 主要研究方向为食品安全检测。E-mail:

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注: A. 1.00%浓度; B. 0.10%浓度; C. 0.01%浓度。

, figureFileSmall=ZJMBWh+lXyAEmAWyrHvqjQ==, figureFileBig=TWTsBtThGBp5U25dZeSr3w==, tableContent=null), ArticleFig(id=1171733753861808475, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153433739548942903, language=EN, label=Fig.2, caption=Results of specificity test, figureFileSmall=Lc5PN7rTNLybklgCIHNC0Q==, figureFileBig=f7UmlkgN/suOrPjYN2HclQ==, tableContent=null), ArticleFig(id=1171733753945694557, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153433739548942903, language=CN, label=图2, caption=特异性测定结果, figureFileSmall=Lc5PN7rTNLybklgCIHNC0Q==, figureFileBig=f7UmlkgN/suOrPjYN2HclQ==, tableContent=null), ArticleFig(id=1171733754008609119, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153433739548942903, language=EN, label=Table 1, caption=

Sensitivity test results of reagent kit

, figureFileSmall=null, figureFileBig=null, tableContent=
检测通道 检测物种 阳性次数/实验次数
1.00%浓度 0.10%浓度 0.01%浓度
猪 猪 15/15 15/15 15/15
牛 牛 15/15 15/15 0/15
羊 羊 15/15 15/15 15/15
鸡 鸡 15/15 15/15 0/15
鸭 鸭 15/15 15/15 0/15
鹅 鹅 15/15 15/15 0/15
马 马 15/15 15/15 15/15
驴 驴 15/15 15/15 15/15
), ArticleFig(id=1171733754113466721, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153433739548942903, language=CN, label=表1, caption=

试剂盒灵敏度检测结果

, figureFileSmall=null, figureFileBig=null, tableContent=
检测通道 检测物种 阳性次数/实验次数
1.00%浓度 0.10%浓度 0.01%浓度
猪 猪 15/15 15/15 15/15
牛 牛 15/15 15/15 0/15
羊 羊 15/15 15/15 15/15
鸡 鸡 15/15 15/15 0/15
鸭 鸭 15/15 15/15 0/15
鹅 鹅 15/15 15/15 0/15
马 马 15/15 15/15 15/15
驴 驴 15/15 15/15 15/15
), ArticleFig(id=1171733754226712931, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153433739548942903, language=EN, label=Table 2, caption=

Real time fluorescence PCR sensitivity detection results

, figureFileSmall=null, figureFileBig=null, tableContent=
检测物种 CT值
1.00%浓度 0.10%浓度 0.01%浓度
猪 19.98 23.38 26.66
牛 23.83 25.25 27.13
羊 19.77 23.34 25.52
鸡 16.44 19.72 22.33
鸭 18.32 20.69 23.18
鹅 19.69 21.57 25.03
马 25.62 27.86 30.55
驴 19.95 22.78 26.48
), ArticleFig(id=1171733754335764837, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153433739548942903, language=CN, label=表2, caption=

实时荧光PCR灵敏度检测结果

, figureFileSmall=null, figureFileBig=null, tableContent=
检测物种 CT值
1.00%浓度 0.10%浓度 0.01%浓度
猪 19.98 23.38 26.66
牛 23.83 25.25 27.13
羊 19.77 23.34 25.52
鸡 16.44 19.72 22.33
鸭 18.32 20.69 23.18
鹅 19.69 21.57 25.03
马 25.62 27.86 30.55
驴 19.95 22.78 26.48
), ArticleFig(id=1171733754428039526, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153433739548942903, language=EN, label=Table 3, caption=

Specificity test results

, figureFileSmall=null, figureFileBig=null, tableContent=
检测通道 检测物种 交叉反应物种
猪 牛、羊、鸡、鸭、鹅、马、驴 无交叉
牛 猪、羊、鸡、鸭、鹅、马、驴 无交叉
羊 猪、牛、鸡、鸭、鹅、马、驴 无交叉
鸡 猪、牛、羊、鸭、鹅、马、驴 无交叉
鸭 猪、牛、羊、鸡、鹅、马、驴 无交叉
鹅 猪、牛、羊、鸡、鸭、马、驴 无交叉
马 猪、牛、羊、鸡、鸭、鹅、驴 无交叉
驴 猪、牛、羊、鸡、鸭、鹅、马 无交叉
), ArticleFig(id=1171733754528702823, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153433739548942903, language=CN, label=表3, caption=

特异性测定结果

, figureFileSmall=null, figureFileBig=null, tableContent=
检测通道 检测物种 交叉反应物种
猪 牛、羊、鸡、鸭、鹅、马、驴 无交叉
牛 猪、羊、鸡、鸭、鹅、马、驴 无交叉
羊 猪、牛、鸡、鸭、鹅、马、驴 无交叉
鸡 猪、牛、羊、鸭、鹅、马、驴 无交叉
鸭 猪、牛、羊、鸡、鹅、马、驴 无交叉
鹅 猪、牛、羊、鸡、鸭、马、驴 无交叉
马 猪、牛、羊、鸡、鸭、鹅、驴 无交叉
驴 猪、牛、羊、鸡、鸭、鹅、马 无交叉
), ArticleFig(id=1171733754587423080, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153433739548942903, language=EN, label=Table 4, caption=

Results of Adulteration simulation test

, figureFileSmall=null, figureFileBig=null, tableContent=
掺入物种 阳性次数/实验次数
基质物种 基质物种组织+1.0%掺入物种组织 基质物种组织+0.5%掺入物种组织
鸭、猪 牛(30/30) 鸭(15/15)+猪(15/15) 鸭(15/15)+猪(15/15)
鸭、猪 羊(30/30) 鸭(15/15)+猪(15/15) 鸭(15/15)+猪(15/15)
鸭、猪 鸡(30/30) 鸭(15/15)+猪(15/15) 鸭(15/15)+猪(15/15)
鸭、猪 鹅(30/30) 鸭(15/15)+猪(15/15) 鸭(15/15)+猪(15/15)
鸭、猪 马(20/20) 鸭(15/15)+猪(15/15) 鸭(15/15)+猪(15/15)
鸭、猪 驴(30/30) 鸭(15/15)+猪(15/15) 鸭(15/15)+猪(15/15)
), ArticleFig(id=1171733754675503465, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153433739548942903, language=CN, label=表4, caption=

掺假模拟检测结果

, figureFileSmall=null, figureFileBig=null, tableContent=
掺入物种 阳性次数/实验次数
基质物种 基质物种组织+1.0%掺入物种组织 基质物种组织+0.5%掺入物种组织
鸭、猪 牛(30/30) 鸭(15/15)+猪(15/15) 鸭(15/15)+猪(15/15)
鸭、猪 羊(30/30) 鸭(15/15)+猪(15/15) 鸭(15/15)+猪(15/15)
鸭、猪 鸡(30/30) 鸭(15/15)+猪(15/15) 鸭(15/15)+猪(15/15)
鸭、猪 鹅(30/30) 鸭(15/15)+猪(15/15) 鸭(15/15)+猪(15/15)
鸭、猪 马(20/20) 鸭(15/15)+猪(15/15) 鸭(15/15)+猪(15/15)
鸭、猪 驴(30/30) 鸭(15/15)+猪(15/15) 鸭(15/15)+猪(15/15)
), ArticleFig(id=1171733754742612330, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153433739548942903, language=EN, label=Table 5, caption=

Results of Adulterated simulation measured QPCR

, figureFileSmall=null, figureFileBig=null, tableContent=
掺入物种 CT值
基质物种 基质物种组织+1.0%掺入物种组织 基质物种组织+0.5%掺入物种组织
鸭、猪 牛(26.48) 鸭(28.45)+猪(29.86) 鸭(33.31)+猪(33.93)
鸭、猪 羊(22.63) 鸭(21.46)+猪(23.92) 鸭(28.56)+猪(29.27)
鸭、猪 鸡(18.01) 鸭(21.63)+猪(22.86) 鸭(24.66)+猪(26.17)
鸭、猪 鹅(19.12) 鸭(22.91)+猪(24.19) 鸭(27.33)+猪(28.97)
鸭、猪 马(24.33) 鸭(23.05)+猪(23.74) 鸭(28.09)+猪(28.69)
鸭、猪 驴(23.68) 鸭(21.93)+猪(23.52) 鸭(27.84)+猪(29.63)
), ArticleFig(id=1171733754860052843, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153433739548942903, language=CN, label=表5, caption=

掺假模拟测QPCR结果

, figureFileSmall=null, figureFileBig=null, tableContent=
掺入物种 CT值
基质物种 基质物种组织+1.0%掺入物种组织 基质物种组织+0.5%掺入物种组织
鸭、猪 牛(26.48) 鸭(28.45)+猪(29.86) 鸭(33.31)+猪(33.93)
鸭、猪 羊(22.63) 鸭(21.46)+猪(23.92) 鸭(28.56)+猪(29.27)
鸭、猪 鸡(18.01) 鸭(21.63)+猪(22.86) 鸭(24.66)+猪(26.17)
鸭、猪 鹅(19.12) 鸭(22.91)+猪(24.19) 鸭(27.33)+猪(28.97)
鸭、猪 马(24.33) 鸭(23.05)+猪(23.74) 鸭(28.09)+猪(28.69)
鸭、猪 驴(23.68) 鸭(21.93)+猪(23.52) 鸭(27.84)+猪(29.63)
), ArticleFig(id=1171733754960716140, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153433739548942903, language=EN, label=Table 6, caption=

False positive rate and false negative rate (%)

, figureFileSmall=null, figureFileBig=null, tableContent=
性能指标 0%浓度 1.00%浓度 0.10%浓度 0.01%浓度
假阴性率 0 0 0 17
假阳性率 0 0 0 0
), ArticleFig(id=1171733755094933869, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153433739548942903, language=CN, label=表6, caption=

假阳性率、假阴性率(%)

, figureFileSmall=null, figureFileBig=null, tableContent=
性能指标 0%浓度 1.00%浓度 0.10%浓度 0.01%浓度
假阴性率 0 0 0 17
假阳性率 0 0 0 0
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应用胶体金免疫层析法检测肉类中多种动物源性成分
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姜玉榕 1 , 马丽 2 , 张维 2 , 李青飞 2 , 栾军 2, *
食品安全质量检测学报 | 食品分析与检测 2025,16(8): 261-266
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食品安全质量检测学报 | 食品分析与检测 2025, 16(8): 261-266
应用胶体金免疫层析法检测肉类中多种动物源性成分
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姜玉榕1 , 马丽2, 张维2, 李青飞2, 栾军2, *
作者信息
  • 1.国家轻工业食品质量监督检测南京站, 南京 210024
  • 2.南京海关动植物与食品检测中心, 南京 210019

    • 姜玉榕(1997—), 女, 主要研究方向为食品安全检测。E-mail:

通讯作者:

* 栾军(1982—), 男, 硕士, 工程师, 主要研究方向为食品安全检测。E-mail:
Application of colloidal gold immunochromatography for detecting various animal derived components in meat
Yu-Rong JIANG1 , Li MA2, Wei ZHANG2, Qing-Fei LI2, Jun LUAN2, *
Affiliations
  • 1. National Light Industry Food Quality Supervision and Inspection Nanjing Station, Nanjing 210024, China
  • 2. Animal, Plant and Food Inspection Center, Nanjing Customs, Nanjing 210019, China
出版时间: 2025-04-25 doi: 10.19812/j.cnki.jfsq11-5956/ts.20241130002
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摘要
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目的 研究胶体金免疫层析法检测肉制品中动物源性成分的准确性及可行性。方法 利用该快速检测试剂盒检测肉制品中动物源性成分, 对样品中8种动物源成分的灵敏度、特异性、掺假模拟、假阳性率、假阴性率进行相关实验, 并以实时荧光定量聚合酶链式反应(quantitative real-time polymerase chain reaction, QPCR)为参比对照。结果 该快速检测试剂盒的灵敏度对牛、鸡、鸭、鹅的检出限可达到0.1%, 对猪、羊、马、驴的检出限可达到0.01%; 掺假模拟中猪、牛、羊、鸡、鸭、鹅、马、驴的检出限均可达到0.5%, 同时与其他非本物种通道检测时均无交叉反应, 特异性较好。结论 本研究建立的胶体金免疫层析法适用于肉制品中动物源性的现场快速筛查和检测。

胶体金免疫层析  /  快速检测  /  动物源成分  /  实时荧光定量聚合酶链式反应

Objective To study the accuracy and feasibility of colloidal gold immunochromatography for the detection of animal-derived components in meat products. Methods The rapid detection kit was used to detect the animal-derived components in meat products. The sensitivity, specificity, adulteration simulation, false positive rate and false negative rate of 8 kinds of animal-derived components were tested, and quantitative real-time polymerase chain reaction (QPCR) was used as a reference. Results The limits of detection of the rapid detection kit could reach 0.1% for cattle, chicken, duck and goose, and 0.01% for pig, sheep, horse and donkey. The limits of detection of pig, cattle, sheep, chicken, duck, goose, horse and donkey in the adulterated simulation could reach 0.5%, and there was no cross-reaction with other non-species channel detection, and the specificity was good. Conclusion The colloidal gold immunochromatography method established in this study is suitable for the rapid screening and detection of animal origin in meat products.

colloidal gold immunochromatography  /  rapid detection  /  animal derived components  /  quantitative real-time polymerase chain reaction
姜玉榕, 马丽, 张维, 李青飞, 栾军. 应用胶体金免疫层析法检测肉类中多种动物源性成分. 食品安全质量检测学报, 2025 , 16 (8) : 261 -266 . DOI: 10.19812/j.cnki.jfsq11-5956/ts.20241130002
Yu-Rong JIANG, Li MA, Wei ZHANG, Qing-Fei LI, Jun LUAN. Application of colloidal gold immunochromatography for detecting various animal derived components in meat[J]. Journal of Food Safety & Quality, 2025 , 16 (8) : 261 -266 . DOI: 10.19812/j.cnki.jfsq11-5956/ts.20241130002
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0 引言
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肉类是蛋白质的重要来源, 并为人类提供必需的氨基酸、维生素、微量元素以及大量的不饱和脂肪酸[1-3]。据联合国粮食及农业组织报道, 2013年全球肉类产量为3.082亿t, 且每年都在大幅增长, 至2050年肉类供应可养活90亿人[4-5], 可见肉制品的质量控制在其生产过程中至关重要。然而当前掺杂掺假、以次充好等牟取暴利的事件仍频繁出现, 从欧洲的“马肉风波”到国内的“驴肉掺假”等[6-8], 不仅严重损害消费者身心健康, 同时也引起了公众对于食品安全的担忧。因此, 建立快速有效的动物源性成分检测方法, 实现对肉类的高通量精准鉴别和对肉制品行业监管具有重要意义[9-10]。
传统的感官鉴别方法主要是通过眼或显微镜对肉类组织结构进行鉴别, 但经加工的肉制品组织结构被破坏, 同时受限于检测人员的经验误差, 会直接影响鉴定结果[11-13]。KUMARI等[14]利用感官评价比较动物肉和植物肉在颜色、质地、味道上的差异。但在利益驱动下不法商贩通过添加香精香料改变肉的色泽、气味等, 以致消费者无法辨别动物源性食品的真实性[15]。基于蛋白质组分发展的酶联免疫吸附法(enzyme-linked immunosorbent, ELISA)、色谱法和质谱法等[16]检测方法, 同样会破坏肉制品本身的组织结构, 且检测过程烦琐, 不利于大规模推广。例如, ZHANG等[17]利用串联质谱法中多反应监测模式(multiple reaction monitoring, MRM)其离子源为电喷雾电离源正离子扫描模式, 在毛细管电压与温度分别为3500 V、275 ℃, 碰撞气压与流速分别为1.5 mTorr、38 Arb的条件下能识别不同动物源性的特异性肽, 并成功鉴别出猪、牛、羊、鹿、鸡、鸭和火鸡动物源性成分。而基于无损伤样品的检测技术, 如生物传感器、红外光谱法等, 虽然避免了对样品本身的破坏, 但所需的检测设备价格较昂贵, 同样不利于推广[18-19]。MABOOD等[20]建立了结合近红外光谱与多变量分析的检测方法, 在包含39种肉类5952份混合样本中添加不同比例的猪肉, 利用傅里叶变换近红外光谱(Fourier transform-near infrared spectroscopy, FT-NIR)在光谱波长10000~4000 cm-1、分辨率2 cm-1、总径0.5 mm的条件下成功分析出纯猪肉与添加猪肉的混合样品之间的区别。基于生物学为基础的检测方法高速发展, 例如, 聚合酶链式反应技术、实时荧光定量聚合酶链式反应(quantitative real-time polymerase chain reaction, QPCR)技术、多重聚合酶链式反应(multiplex polymerase chain reaction, MPCR)技术等, IZADPANAH等[21]利用多重反应PCR通过在细胞色素C氧化酶亚基(COX1)基因保守区设计特异性引物, 成功区分猪、骆驼、绵羊、驴、山羊、牛以及鸡的动物源性成分。当前, 这类检测方法广泛应用于肉制品中动物源性检测中, 但仍受限于检测周期长、成本高, 因此不适用于大规模样品的快速筛查。而基于抗原抗体特异性免疫结合反应原理的胶体金免疫层析法, 相比较上述几种类型的检测方法具有操作简单、重现性好、成本低等优势[22-26], 近年来被广泛应用于食品中药物残留检测, 如陈文硕等[27]通过建立胶体金免疫层析法成功检测出动物源性食品中诺佛沙星; 纪丽君等[28]利用胶体金免疫层析法成功检测出山药中咪鲜胺。但目前应用胶体金免疫层析法检测肉制品中动物源性成分的研究鲜有报道。
本研究基于胶体金免疫层析法的原理, 采用由杭州泰熙生物技术有限公司提供的动物源性快速检测试剂盒, 对市售的肉制品进行检测。按照国家市场监督管理局《总局关于规范食品快速检测方法使用管理的意见》对该方法的检出线、假阳性率、假阴性率、特异性以及参比方法的一致性进行评价[29-31]。本方法的使用可以为基层监管部门对肉制品中动物源性掺假成分鉴定提供可靠的技术支撑。
1 材料与方法
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1.1 材料与试剂
实验用猪肉、牛肉、羊肉、鸡肉、鸭肉、鹅肉、马肉、驴肉均购于农贸市场、超市。
2×MASTER MIX (H20240105)(上海辉睿生物科技有限公司); Real Time PCR Bovine DNA Detection Kit (AN81384A)、Real Time PCR Ovine DNA Detection Ki (AM82588A)、Real Time PCR Porcine DNA Detection Kit (AN70800A)[宝日医生物技术(北京)有限公司]; 动物源性成分(猪、牛、羊、鸡、鸭、鹅、马、驴)快速检测试剂盒(杭州泰熙生物技术有限公司)。
1.2 仪器与设备
JA31002分析天平(精度0.01 mg, 上海精密仪器仪表有限公司); Sorvall ST16高速离心机、ViiA7 ABI荧光定量PCR仪、QuantStudio5实时荧光定量PCR仪(美国赛默飞世尔科技公司); KZ-II样品研磨机(上海净信实业发展有限公司)。
1.3 实验方法
QPCR检测肉制品中动物源性成分依据如下: 牛、羊、猪成分检测依据SN/T 2051—2008《食品、化妆品和饲料中牛羊猪源性成分检测方法 实时PCR法》, 鸡、鸭、鹅成分检测依据GB/T 38164—2019《常见畜禽动物源性成分检测方法 实时荧光PCR法》, 马成分检测依据SN/T 3730.5—2013《食品及饲料中常见畜类品种的鉴定方法 第5部分: 马成分检测实时荧光PCR法》, 驴成分检测依据SN/T 3730.4—2013《食品及饲料中常见畜类品种的鉴定方法 第4部分: 驴成分检测实时荧光PCR法》。
1.3.1 灵敏度检测
鸡鸭等肉制品去皮、去脂, 取组织均质, 称1 g试样置于25 mL离心管中, 加入10 mL样本稀释液, 匀浆、离心(8000 r/min, 5 min), 取上层清液配制成浓度梯度为1.00%、0.10%、0.01%的待测液, 吸取100 μL待测液滴加到试纸条的加样口, 静置5~10 min, 观察颜色变化。质控线(C线)和检测线(T线)均出现红色为阴性, 仅C线出现红色为阳性、C线不显色为试纸条无效。8种动物源性成分设置3种不同浓度梯度, 每种浓度各5份, 共计120份, 每份样品重复检测3次。根据实验结果确定试剂条的检出限。
1.3.2 特异性检测
选择猪、牛、羊、鸡、鸭、鹅、马、驴为物种样本, 使用棉拭子吸取样本组织液, 用试纸条检测5~10 min, 观察颜色变化, 每种物种样品各4份, 共计32份, 每份样品重复测定3次, 根据结果确定试剂条的特异性。
1.3.3 掺假模拟
分别称取9.8 g基质物种(牛、羊、鸡、鹅、马、驴)与0.1 g掺入物种(鸭、猪), 加入到10 mL稀释液振荡混匀, 配制成1%掺入模拟样品待测液, 取10 g基质物种配制成相同浓度的基质物种稀释液, 将掺入模拟样品待测液与基质物种稀释液等体积混匀成0.5%掺入模拟样品待测液, 吸取100 μL待测液滴加到试纸条的加样口, 静置5~10 min, 并观察颜色变化。6种基质物种配制成2种浓度梯度, 每种浓度各5份, 共计60份, 每份样品重复检测3次。
1.3.4 假阳性率、假阴性率测定
按照1.3.1前处理过程制备样品, 将8种动物源性成分设置3种不同浓度梯度, 每种浓度各5份, 共计120份, 每份样品重复测定3次, 并以空白样品为对照。按公式(1)~(3)分别计算假阳性率、假阴性率。
灵敏度/%=$\frac{{{C}_{0}}}{{{C}_{1}}}$×100%
假阴性率/%=$\frac{{{C}_{3}}}{{{C}_{1}}}$×100%
假阳性率/%=$\frac{{{C}_{4}}}{{{C}_{2}}}$×100%
式中: C0为检出阳性结果的阳性样品数; C1为总阳性样品数; C2为总阴性数; C3为阳性样品的阴性结果数; C4为阴性样品的阳性结果数。
1.3.5 参比对照实验
相同条件下处理样品, 并按照上述1.3中QPCR法对样品进行同步测定, 比较两种方法测定结果的符合率。
1.4 数据处理
采用Excel 2019对数据进行处理。
2 结果与分析
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2.1 猪、牛、羊、鸡、鸭、鹅、马、驴灵敏度检验结果分析
本研究使用的动物源性成分快速检测试剂盒检测结果如表1所示, 对猪、羊、马、驴的检出限可达到0.01%; 对牛、鸡、鹅、鸭的检出限可达到0.10%。如图1所示, 当试样中马、驴含量为0.01%、0.10%、1.00%时, C线颜色明显深于T线, 检测结果为阳性, 且平行实验间重复性好。但牛、鸡、鸭、鹅4种肉源性试样浓度为0.01%时, 该试剂盒未检出阳性, 表明该试剂盒存在一定的局限性, 仅能对特定的几种动物源性具有较高的检出限。
为进一步验证该动物源性成分快速检测试剂盒的灵敏度, 以QPCR检测为参比对照, 当8种动物源成分检测结果的循环阀(cycle threshold, CT)≤35时, 表明该种动物源性成分检出。反之, 则未检出。结果如表2所示, 当8种动物源性成分浓度分别为1.00%、0.10%时, 所有试样的CT值均小于35, 8种动物源性成分均可检出, 这与该试剂盒的检测结果相一致, 当检测浓度为0.01%时, 所有试样CT值均小于35, 表明仍可检出, 而本试剂盒仅能检出猪、羊、马、驴4种动物源性成分, 可见当浓度为0.01%时, 本试剂盒检测结果可靠性与QPCR检测有一定差距。
2.2 猪、牛、羊、鸡、鸭、鹅、马、驴特异性检验结果分析
如图2所示, 当检测通道为牛时检测结果为两条线(阴性), 此时其他物种检测结果均显示一条线(阳性), 即该试剂盒对猪、牛、羊、鸡、鸭、鹅、马、驴物种进行其他非本物种通道检测时均无交叉反应, 检测结果与表3相符, 表明该试剂盒具有较高的特异性。
2.3 猪、牛、羊、鸡、鸭、鹅、马、驴模拟掺假检测结果分析
由表4可知, 当基质物种(牛、羊、鸡、鹅、马、驴)分别与掺入物种(鸭、猪)混成1.0%和0.5%两种浓度时, 该试剂盒检测结果均显示为阳性。同时, 采用QPCR为参比对照时, 结果如表5可知, 所有样本检测结果的CT值均小于35, 表明所有检测样本中均能检测出猪、鸭两种动物源成分, 这与本研究所用试剂盒检测结果相一致。表明该试剂盒在模拟掺假测试中的检出限达到了0.5%。
2.4 猪、牛、羊、鸡、鸭、鹅、马、驴假阳性率与假阴性率结果分析
由表6可知, 当样品浓度为1.00%和0.10%时, 假阳性率与假阴性率均为0, 当样品浓度为0.01%时, 假阴性率为17%。可能是当样本提取液浓度过低时, 溶液中存在少量杂质, 使得该试剂盒无法检出阳性。
3 结论
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肉制品掺假是食品安全领域一直存在的问题, 针对肉源性成分鉴定, 本研究建立了一种能够同时检测肉制品中8种动物源性成分的胶体金免疫层析法, 并对该方法进行验证。结果表明该检测方法对牛、鸡、鸭、鹅的检出限可达到0.10%, 对猪、羊、驴、马的检出限可达到0.01%; 掺假模拟实验中对猪、牛、羊、鸡、鸭、鹅、马、驴的检出限均可达到0.5%。根据特异性实验结果可以看出本研究使用的快速检测试剂盒在检测8种动物源成分与其他非本物种通道时均无交叉反应, 具有较好的特异性。结合实时荧光定量PCR检测结果, 两者具有较好的一致性。但当样品浓度较低时, 该试剂盒出现较高的假阴性率, 因此, 可以进一步优化样品前处理, 去除更多杂质以提高该试剂盒检测精度。总体来说, 该试剂盒的检测结果准确、可靠, 符合商品化要求。相比较于QPCR技术, 对仪器和试剂的要求低, 是一种低成本的检测技术, 更适用于基层市场的肉制品质量检测。
基金
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  • 南京海关科研项目(2025KJ30)
文献
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2025年第16卷第8期
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doi: 10.19812/j.cnki.jfsq11-5956/ts.20241130002
  • 接收时间：2024-11-30
  • 首发时间：2025-07-19
  • 出版时间：2025-04-25
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  • 收稿日期：2024-11-30
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南京海关科研项目(2025KJ30)
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    1.国家轻工业食品质量监督检测南京站, 南京 210024
    2.南京海关动植物与食品检测中心, 南京 210019

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* 栾军(1982—), 男, 硕士, 工程师, 主要研究方向为食品安全检测。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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