Article(id=1153433742031966226, tenantId=1146029695717560320, journalId=1149652044408987649, issueId=1153433737141412332, articleNumber=null, orderNo=null, doi=10.19812/j.cnki.jfsq11-5956/ts.20241014004, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=null, receivedDate=1728835200000, receivedDateStr=2024-10-14, revisedDate=null, revisedDateStr=null, acceptedDate=null, acceptedDateStr=null, onlineDate=1752929633863, onlineDateStr=2025-07-19, pubDate=1745510400000, pubDateStr=2025-04-25, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1752929633863, onlineIssueDateStr=2025-07-19, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1752929633863, creator=13701087609, updateTime=1752929633863, 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=93, endPage=99, ext={EN=ArticleExt(id=1153433742581420053, articleId=1153433742031966226, tenantId=1146029695717560320, journalId=1149652044408987649, language=EN, title=Effection and comparison of detection results of aflatoxin B1 in foods by fully automated immunomagnetic bead purification method and immunoaffinity column method, columnId=1151895321526759957, journalTitle=Journal of Food Safety & Quality, columnName=Special Topic: Detection and Prevention and Control of Biotoxins in Food, runingTitle=null, highlight=null, articleAbstract=

Objective To compare the detection results of aflatoxin B1 (AFB1) content in various matrices such as rice, wheat products, and peanut products using 2 kinds of different pretreatment methods: Fully automated immunomagentic bead purification and immunoaffinity column pretreatment method. Methods After extraction with methanol:water (70:30, V:V), a portion of the extract was purified using the immunoaffinity column method with an AFB1-specific immunoaffinity column, while another portion of the extract was processed by a fully automated immunomagnetic bead purification system paired with an immunomagnetic bead kit. Finally, both purified extracts were analyzed using high performance liquid chromatography coupled with post-column derivatization for detection. Results The efficacy of the immunomagetic bead purification kit and the column efficiency of the immunoaffinity column were both greater than 95%, which could meet the experimental requirements. Within mass concentration range of 1.00-50.00 ng/mL, the linear correlation coefficient (r) was 0.99993, indicating a good linear relationship. The 3 level spiked recovery experiments were conducted in rice matrix (n=6), and the recovery rates of the 2 kinds of pretreatment methods ranged from 98.2%-106.0%, with a relative standard deviation range of 1.0%-5.0%; single level spiked recovery experiments were conducted in wheat products (n=6), with a recovery rate exceeding 80% and a relative standard deviation of 3.7%-7.2%. Meanwhile, the 2 kinds of methods were used to detected AFB1 positive peanut products and the quality control sample of corn flour matrix, their results were subjected to t-test, which showed no significant difference. Further, Bland-Altman statistical analysis was carried out on the results obtained from the quality control samples of corn flour matrix after 2 kinds of pretreatment methods were used. The results showed that the 2 kinds of methods could replace each other in the detection of AFB1 in this matrix. The purification step of the immunoaffinity column method was time-consuming, 20 batches of samples take about 3-4 hours. And could only be manually operated, which generated about 1000 mL waste liquid; while the immunomagetic bead method used a fully automatic magnetic bead purifier, which automatically completed 20 batches of samples in 40 minutes at one time, with almost no waste liquid generated. Conclusion The fully automated immunomagnetic bead purification method and the immunoaffinity column method both can achieve good results in detecting AFB1 in various matrices. Also, the immunomagnetic bead purification method is more efficient, easy to handle, generates less waste liquid, and the cost can be reduced by about 50%.

, correspAuthors=Chun-Hong SHI, 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=Qing ZHU, Mei-Ping CAO, Xing-Fa REN, Rui CHEN, Feng-Wei SHAO, Gui-Xia HU, Chun-Hong SHI), CN=ArticleExt(id=1153433755210469615, articleId=1153433742031966226, tenantId=1146029695717560320, journalId=1149652044408987649, language=CN, title=全自动免疫磁珠纯化法与免疫亲和柱法对食品中黄曲霉毒素B1检测结果的影响比对, columnId=1151895321669366295, journalTitle=食品安全质量检测学报, columnName=本期专题:食品中生物毒素检测与防控, runingTitle=null, highlight=null, articleAbstract=

目的 采用全自动免疫磁珠纯化法与免疫亲和柱法对大米、小麦制品、花生制品等多种基质进行前处理, 检测其黄曲霉毒素B1 (aflatoxin, AFB1)含量, 并比较两种前处理方法的检测结果。方法 样品经甲醇:水(70:30, V:V)提取后, 一部分提取液采用免疫亲和柱法经AFB1专用免疫亲和柱净化, 另取一部分提取液采用搭配免疫磁珠试剂盒的全自动免疫磁珠纯化仪进行前处理, 最后都采用高效液相色谱法-柱后衍生法进行检测。结果 免疫磁珠纯化试剂盒效能和免疫亲和柱柱效均大于95%, 皆能满足实验要求。在质量浓度为1.00~50.00 ng/mL的线性范围内, 线性相关系数(r)为0.99993, 线性关系良好。在大米基质中进行3水平加标回收率实验(n=6), 两种前处理方法回收率范围为98.2%~106.0%, 相对标准偏差为1.0%~5.0%; 在小麦制品中进行单水平加标回收率实验(n=6), 回收率超过80%, 相对标准偏差为3.7%~7.2%。AFB1阳性花生制品、玉米粉基质质控样品采用两种前处理方法进行检测, 并对结果进行t检验, 显示所得结果无显著性差异; 对玉米粉基质质控样品采用两种前处理方法进行检测后所得结果进行Bland-Altman统计分析, 结果表明在检测该基质AFB1时两种方法可以相互替代。免疫亲和柱法净化步骤耗时长, 20批样品耗时约3~4 h; 只能人工操作, 产生大约1000 mL废液; 而免疫磁珠法采用全自动磁珠纯化仪, 净化步骤一次40 min, 可自动完成20批样品, 几乎无废液产生。结论 全自动免疫磁珠纯化法与免疫亲和柱法检测多种基质中的AFB1均能取得良好效果, 且免疫磁珠纯化法效率高、处理简便、产生废液少, 成本可降低大约50%。

, correspAuthors=石春红, authorNote=null, correspAuthorsNote=
* 石春红(1969—), 女, 硕士, 正高级工程师, 主要研究方向为食品检验质量控制及管理。E-mail:
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朱青(1988—), 女, 硕士, 中级检验师, 主要研究方向为食品质量与安全。E-mail:

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朱青(1988—), 女, 硕士, 中级检验师, 主要研究方向为食品质量与安全。E-mail:

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Recoveries and precisions tests of AFB1 in rice matrix (n=6)

, figureFileSmall=null, figureFileBig=null, tableContent=
方法 加标浓度/(μg/kg) 平均回收率/% RSDs/%
IMB法 5.0 99.3 4.21
10.0 98.2 4.93
30.0 106.0 2.56
IAC法 5.0 102.0 2.50
10.0 102.0 1.03
30.0 99.4 1.46
), ArticleFig(id=1171733749466177787, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153433742031966226, language=CN, label=表1, caption=

大米中AFB1加标回收率及精密度实验(n=6)

, figureFileSmall=null, figureFileBig=null, tableContent=
方法 加标浓度/(μg/kg) 平均回收率/% RSDs/%
IMB法 5.0 99.3 4.21
10.0 98.2 4.93
30.0 106.0 2.56
IAC法 5.0 102.0 2.50
10.0 102.0 1.03
30.0 99.4 1.46
), ArticleFig(id=1171733749529092348, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153433742031966226, language=EN, label=Table 2, caption=

Intra-day precisions of IMB method

, figureFileSmall=null, figureFileBig=null, tableContent=
加标回收率1(第1 d) 加标回收率2(第2 d) 加标回收率3(第3 d)
测定值/% 平均值/% RSDs/% 测定值/% 平均值/% RSDs/% 测定值/% 平均值/% RSD/%
89.0 88.5 0.51 84.6 83.9 0.73 90.4 92.0 1.99
88.1 83.4 91.6
88.5 83.8 94.0
), ArticleFig(id=1171733749583618301, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153433742031966226, language=CN, label=表2, caption=

IMB法日间精密度

, figureFileSmall=null, figureFileBig=null, tableContent=
加标回收率1(第1 d) 加标回收率2(第2 d) 加标回收率3(第3 d)
测定值/% 平均值/% RSDs/% 测定值/% 平均值/% RSDs/% 测定值/% 平均值/% RSD/%
89.0 88.5 0.51 84.6 83.9 0.73 90.4 92.0 1.99
88.1 83.4 91.6
88.5 83.8 94.0
), ArticleFig(id=1171733749650727166, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153433742031966226, language=EN, label=Table 3, caption=

Detecting results of AFB1 in the quality control samples by 2 kinds of methods

, figureFileSmall=null, figureFileBig=null, tableContent=
货号 标准值/(μg/kg) 特性值区间
/(μg/kg)
方法
名称
测定值/(μg/kg) 平均值 RSDs
/%
准确度
/%
1 2 3 4 5 6 /(μg/kg)
中国检科院QC-WM-705 38.91 30.33~47.49 IMB 35.68 35.48 33.79 33.57 35.92 35.81 35.04 3.05 -9.9
IAC 33.66 33.14 34.50 34.27 34.98 36.29 34.47 3.19 -11.4
), ArticleFig(id=1171733749717836031, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153433742031966226, language=CN, label=表3, caption=

两种方法质控样品检测结果

, figureFileSmall=null, figureFileBig=null, tableContent=
货号 标准值/(μg/kg) 特性值区间
/(μg/kg)
方法
名称
测定值/(μg/kg) 平均值 RSDs
/%
准确度
/%
1 2 3 4 5 6 /(μg/kg)
中国检科院QC-WM-705 38.91 30.33~47.49 IMB 35.68 35.48 33.79 33.57 35.92 35.81 35.04 3.05 -9.9
IAC 33.66 33.14 34.50 34.27 34.98 36.29 34.47 3.19 -11.4
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全自动免疫磁珠纯化法与免疫亲和柱法对食品中黄曲霉毒素B1检测结果的影响比对
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朱青 1 , 曹美萍 1 , 任兴发 2, 3 , 陈睿 1 , 邵锋伟 2 , 胡桂霞 1 , 石春红 1, *
食品安全质量检测学报 | 本期专题:食品中生物毒素检测与防控 2025,16(8): 93-99
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食品安全质量检测学报 | 本期专题:食品中生物毒素检测与防控 2025, 16(8): 93-99
全自动免疫磁珠纯化法与免疫亲和柱法对食品中黄曲霉毒素B1检测结果的影响比对
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朱青1 , 曹美萍1, 任兴发2, 3, 陈睿1, 邵锋伟2, 胡桂霞1, 石春红1, *
作者信息
  • 1.上海市松江食品药品检验所, 上海 201306
  • 2.月旭科技(上海)股份有限公司, 上海 201613
  • 3.上海理工大学, 上海食品快速检测工程技术研究中心, 上海 200093
  • 朱青(1988—), 女, 硕士, 中级检验师, 主要研究方向为食品质量与安全。E-mail:

通讯作者:

* 石春红(1969—), 女, 硕士, 正高级工程师, 主要研究方向为食品检验质量控制及管理。E-mail:
Effection and comparison of detection results of aflatoxin B1 in foods by fully automated immunomagnetic bead purification method and immunoaffinity column method
Qing ZHU1 , Mei-Ping CAO1, Xing-Fa REN2, 3, Rui CHEN1, Feng-Wei SHAO2, Gui-Xia HU1, Chun-Hong SHI1, *
Affiliations
  • 1. Shanghai Songjiang Institute for Food and Drug Control, Shanghai 201306, China
  • 2. Welch Materials (Shanghai), Inc., Shanghai 201613, China
  • 3. Shanghai Engineering Research Center of Food Rapid Detection, University of Shanghai for Science and Technology, Shanghai 200093, China
出版时间: 2025-04-25 doi: 10.19812/j.cnki.jfsq11-5956/ts.20241014004
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目的 采用全自动免疫磁珠纯化法与免疫亲和柱法对大米、小麦制品、花生制品等多种基质进行前处理, 检测其黄曲霉毒素B1 (aflatoxin, AFB1)含量, 并比较两种前处理方法的检测结果。方法 样品经甲醇:水(70:30, V:V)提取后, 一部分提取液采用免疫亲和柱法经AFB1专用免疫亲和柱净化, 另取一部分提取液采用搭配免疫磁珠试剂盒的全自动免疫磁珠纯化仪进行前处理, 最后都采用高效液相色谱法-柱后衍生法进行检测。结果 免疫磁珠纯化试剂盒效能和免疫亲和柱柱效均大于95%, 皆能满足实验要求。在质量浓度为1.00~50.00 ng/mL的线性范围内, 线性相关系数(r)为0.99993, 线性关系良好。在大米基质中进行3水平加标回收率实验(n=6), 两种前处理方法回收率范围为98.2%~106.0%, 相对标准偏差为1.0%~5.0%; 在小麦制品中进行单水平加标回收率实验(n=6), 回收率超过80%, 相对标准偏差为3.7%~7.2%。AFB1阳性花生制品、玉米粉基质质控样品采用两种前处理方法进行检测, 并对结果进行t检验, 显示所得结果无显著性差异; 对玉米粉基质质控样品采用两种前处理方法进行检测后所得结果进行Bland-Altman统计分析, 结果表明在检测该基质AFB1时两种方法可以相互替代。免疫亲和柱法净化步骤耗时长, 20批样品耗时约3~4 h; 只能人工操作, 产生大约1000 mL废液; 而免疫磁珠法采用全自动磁珠纯化仪, 净化步骤一次40 min, 可自动完成20批样品, 几乎无废液产生。结论 全自动免疫磁珠纯化法与免疫亲和柱法检测多种基质中的AFB1均能取得良好效果, 且免疫磁珠纯化法效率高、处理简便、产生废液少, 成本可降低大约50%。

黄曲霉毒素B1  /  免疫磁珠纯化法  /  免疫亲和柱法  /  高效液相色谱法-柱后衍生法

Objective To compare the detection results of aflatoxin B1 (AFB1) content in various matrices such as rice, wheat products, and peanut products using 2 kinds of different pretreatment methods: Fully automated immunomagentic bead purification and immunoaffinity column pretreatment method. Methods After extraction with methanol:water (70:30, V:V), a portion of the extract was purified using the immunoaffinity column method with an AFB1-specific immunoaffinity column, while another portion of the extract was processed by a fully automated immunomagnetic bead purification system paired with an immunomagnetic bead kit. Finally, both purified extracts were analyzed using high performance liquid chromatography coupled with post-column derivatization for detection. Results The efficacy of the immunomagetic bead purification kit and the column efficiency of the immunoaffinity column were both greater than 95%, which could meet the experimental requirements. Within mass concentration range of 1.00-50.00 ng/mL, the linear correlation coefficient (r) was 0.99993, indicating a good linear relationship. The 3 level spiked recovery experiments were conducted in rice matrix (n=6), and the recovery rates of the 2 kinds of pretreatment methods ranged from 98.2%-106.0%, with a relative standard deviation range of 1.0%-5.0%; single level spiked recovery experiments were conducted in wheat products (n=6), with a recovery rate exceeding 80% and a relative standard deviation of 3.7%-7.2%. Meanwhile, the 2 kinds of methods were used to detected AFB1 positive peanut products and the quality control sample of corn flour matrix, their results were subjected to t-test, which showed no significant difference. Further, Bland-Altman statistical analysis was carried out on the results obtained from the quality control samples of corn flour matrix after 2 kinds of pretreatment methods were used. The results showed that the 2 kinds of methods could replace each other in the detection of AFB1 in this matrix. The purification step of the immunoaffinity column method was time-consuming, 20 batches of samples take about 3-4 hours. And could only be manually operated, which generated about 1000 mL waste liquid; while the immunomagetic bead method used a fully automatic magnetic bead purifier, which automatically completed 20 batches of samples in 40 minutes at one time, with almost no waste liquid generated. Conclusion The fully automated immunomagnetic bead purification method and the immunoaffinity column method both can achieve good results in detecting AFB1 in various matrices. Also, the immunomagnetic bead purification method is more efficient, easy to handle, generates less waste liquid, and the cost can be reduced by about 50%.

aflatoxin B1  /  immunomagnetic bead purification method  /  immunoaffinity column method  /  high performance liquid chromatography-post-column derivatization method
朱青, 曹美萍, 任兴发, 陈睿, 邵锋伟, 胡桂霞, 石春红. 全自动免疫磁珠纯化法与免疫亲和柱法对食品中黄曲霉毒素B1检测结果的影响比对. 食品安全质量检测学报, 2025 , 16 (8) : 93 -99 . DOI: 10.19812/j.cnki.jfsq11-5956/ts.20241014004
Qing ZHU, Mei-Ping CAO, Xing-Fa REN, Rui CHEN, Feng-Wei SHAO, Gui-Xia HU, Chun-Hong SHI. Effection and comparison of detection results of aflatoxin B1 in foods by fully automated immunomagnetic bead purification method and immunoaffinity column method[J]. Journal of Food Safety & Quality, 2025 , 16 (8) : 93 -99 . DOI: 10.19812/j.cnki.jfsq11-5956/ts.20241014004
真菌毒素污染是世界性食品安全问题, 也是我国食品安全监管的重点[1]。世界卫生组织(World health Organization, WHO)和联合国粮农组织(Food and Agriculture Organization of the United Nations, FAO)把真菌毒素列为食源性疾病的3大根源之首。其中, 黄曲霉毒素B1 (aflatoxin, AFB1)因其热稳定性好, 268 ℃以上的高温下才能裂解[2], 对人类具有致癌、致畸、致肝损伤作用[3]; 引起农作物污染及产量降低等严重问题[4-5], 因此WHO将黄曲霉毒素确定为1A类致癌物[6-7]。霉菌处于高温(25~32 ℃)、高湿(90%~110%)环境时易产生霉菌毒素, 且经常在粮食(如稻谷、小麦、花生等作物)及其制品中检测到[8-9]。因此, 对粮食及油料作物中黄曲霉毒素进行检测就显得十分关键。目前, 世界各国(如美国、欧盟、日本等国家)均为其制定了相应的限量标准, 欧盟要求花生、谷物及其制品中AFB1限量不超过2 μg/kg, 美国要求黄曲霉毒素总量(除牛奶外)不超过20 μg/kg[10]。我国GB 2761—2017《食品安全国家标准 食品中真菌毒素限量标准》也对不同食品中AFB1做了相应的限量规定。
目前的黄曲霉毒素定量检测前处理方法主要有免疫亲和柱(immunoaffinity column, IAC)法[11-13]、固相萃取法[14-15]、QuEChERS法[16-17]等, 然而这些方法存在着前处理复杂、自动化水平低、成本高、效率低、需要专业人员操作等问题。我国现行检测黄曲霉毒素的GB 5009.22—2016《食品中黄曲霉毒素B族和G族的测定》, 前三法前处理均采用免疫亲和柱法, 在实践中该方法不仅成本较高, 耗时较长, 且对于较为复杂的基质, 易出现柱子堵塞问题, 影响实验结果, 极大降低实验效率[18]。因此, 亟需开发一种新的效率高、准确度高、精密度良好的方法对黄曲霉毒素类含量进行检测, 以期可以替代或改善原有的方法。近年来, 由于免疫磁珠纯化(immunomagnetic bead purification method, IMB)法净化食品、中药材、生化材料等前处理简单、自动化水平高、便捷高效、结果准确度和精密度良好, 并且可以重复利用等诸多优点, 受到检测人员的欢迎[19-21]。例如文献[21]中IMB法可检测食品中的食源性致病菌、重金属离子以及转基因大豆中的DNA等, 但其产量低、易团聚等也是其遇到瓶颈问题。IMB法在生物化学[22]、蛋白质组学[23]等方面已有成熟研究, 在食品安全特别是真菌毒素方面的研究也受到颇多关注[24-28]
基于此, 本研究采用将AFB1抗体偶联在磁珠上形成的AFB1免疫磁珠制作而成的检测试剂盒, 并结合全自动免疫纯化仪进行检测。免疫磁珠净化方法即通过免疫磁珠上的AFB1抗体特异性的亲和作用将AFB1从样品提取液中识别和富集起来, 清洗杂质, 从而起到富集和净化作用; 再利用有机溶剂破坏免疫磁珠上AFB1抗体的空间结构, 将AFB1从免疫磁珠上洗脱下来, 便于后续收集、分析和检测[29]。仅需将样品提取液放入混合液中, 试剂盒在免疫磁珠纯化仪中自动完成抗体识别、抗原抗体结合、淋洗、洗脱等步骤, 最终得到可进一步检测的纯化液。
本研究按照现行GB 5009.22—2016中的第三法 高效液相色谱法-柱后衍生法进行检测, 采用AFB1免疫磁珠试剂盒与自动化前处理过程相结合制造的免疫磁珠纯化仪, 对不同食品基质中AFB1进行快速批量前处理, 并与免疫亲和柱法前处理所得结果进行比较, 为日常检测工作提供方法参考。
大米、小麦制品(阳春面)及花生制品(花生仁炒货): 市售; 质控样品购自中国检验检疫科学研究院货号QC-WM-705, 质控样品基质为玉米粉。
氯化钠(分析纯, 上海凌峰化学试剂有限公司); 甲醇、乙腈(色谱级, 德国Merck公司); 磷酸盐缓冲溶液(phosphate buffered solutions, PBS)(色谱纯, pH为7.4±0.1, 上海市松江食品药品检验所配制); AFB1标准溶液(质量浓度2.00 μg/mL, 美国ROMER公司)、AFB1&AFB2& AFG1&AFG2混合标准溶液(总质量浓度5.04 μg/mL, 印度BIO PURE公司)。
SECURA224-1CN分析天平(精度0.0001 g, 德国赛多利斯公司); Milli-Q超纯水系统(密理博中国有限公司); Watbule P11全自动磁珠纯化仪、Welch AFB1磁珠纯化试剂盒、Welch Welview型光化学衍生器、Welch Welchrom AFB1免疫亲和柱[月旭科技(上海)股份有限公司]; FM200超离心研磨仪(北京格瑞德曼仪器设备有限公司); Waters Arc高效液相色谱仪(配荧光检测器)(美国Waters公司); Welch Ultimate XB-C18 [0.46 cm×50 mm, 5 μm, 月旭科技(上海)股份有限公司]。
准确称取5 g经过磨细(粒度小于2 mm)的试样, 与20.0 mL样品提取液[甲醇:水(7:3, V:V)]混合; 转移至50 mL离心管中, 振荡提取20~30 min; 4000 r/min离心5 min; 取2 mL上清液注入样品槽中; 打开Watbule P11 全自动磁珠纯化仪样品仓, 把磁棒套安装到磁棒套筒支架上; 把试剂盒托盘拉出, 将试剂盒放到试剂盒托盘上, 将试剂盒托盘再推回到仪器里面, 确保试剂托盘推到位, 关闭样品仓; 点击开始按钮, 仪器开始进行试剂盒扫码, 扫码完成后, 仪器界面上对应的试剂盒位置出现绿色和白色, 为正确状态。仪器按预定程序自动进行样品纯化。运行完毕后, 用纯净水将试剂盒洗脱管中的洗脱液定容至2.0 mL(视AFB1浓度也可定容至1.0 mL), 混匀后经0.22 μm滤膜过滤, 上机检测。
准确称取5 g(精确至0.01 g)粉碎的样品于50 mL离心管中, 精密加入25.00 mL甲醇:水溶液(70:30, V:V)混匀, 加入5.0 g氯化钠以均质器高速搅拌, 提取2 min, 在4000 r/min离心5 min。准确移取2.00 mL滤液并加入20 mL纯净水稀释, 用玻璃纤维滤纸过滤, 玻璃纤维滤纸快速、高载量、液体中颗粒保留1.6 μm。取稀释后液体待测。
净化: 将恢复室温的免疫亲和柱内的液体放弃后, 连接于20 mL注射器下, 将上述提取液注入注射器; 将空气压力泵与注射器连接, 调节压力使溶液以约6 mL/min (1~2滴/s)流速缓慢通过免疫亲和柱, 直至2~3 mL空气通过柱体。以10.0 mL水淋洗柱子两次, 弃去全部流出液, 并使2~3 mL空气通过柱体。准确加入1.0 mL甲醇洗脱, 流速为1~2 mL/min (小于1滴/s), 收集全部洗脱液于玻璃试管中, 用水定容至2.0 mL。混匀后0.22 μm滤膜过滤, 收集滤液于进样瓶, 上机检测。
色谱柱: Welch Ultimate XB-C18 (0.46 cm×50 mm, 5 μm); 柱后连接光化学衍生器; 流动相: A相: 水, B相: 乙腈:甲醇(50:50, V:V)。等度洗脱: A相:B相(25:75, V:V); 流速: 1.0 mL/min; 柱温: 40 ℃; 进样体积: 50 μL; 检测波长: 激发波长 360 nm; 发射波长440 nm。
通过Waters ARC仪器配置工作站系统进行数据采集, 利用Microsoft Office Excel 2007软件、SPSS 24.0软件进行数据分析及图形绘制。95%置信区间内, P<0.05表示有显著性差异。
于样品槽中加入2 mL 70%甲醇水溶液, 加入终浓度为10 ng/mL的AFB1标准溶液, 以下按1.3.1方法进行。经计算试剂盒效能为95.7% (n=3)。
于50 mL具塞离心管中, 加入2 mL 70%甲醇水溶液及约20 mL 水, 加入终质量浓度为10 ng/mL的AFB1标准溶液, 以下按1.3.2方法进行。经计算柱效为95.0% (n=3)。
经考察, 该批免疫磁珠试剂盒效能及该批次免疫亲和柱柱效均符合GB 5009.22—2016中附录B.2 (>80%)的要求, 可以用于进一步实验。
准确移取适量AFB1标准储备溶液制备标准中间溶液, 用流动相配制相应浓度的标准曲线工作溶液。上机后, 以AFB1浓度为横坐标, 峰面积为纵坐标得标准曲线, 结果表明, 在1.00~50.00 ng/mL AFB1质量浓度范围内, 峰面积(Y)与质量浓度(X, ng/mL)呈良好的线性关系, 线性方程为Y=2.22e+0.06X。线性相关系数r为0.99993。
IAC法定量限(limit of quantitation, LOQ)按照GB 5009.22—2016实验后取得, 确认信噪比(signal/noise, S/N)大于10。IMB法检出限(limit of detection, LOD)、LOQ用稀释法取得, 以S/N大于3作为LOD, S/N大于10作为LOQ。两种方法可达到相同的LOQ (0.10 μg/kg); IMB法可达到GB 5009.22—2016规定的LOD (0.03 μg/kg); IAC的LOD未进行检测。
与对照品溶液相比, 两种方法对于大米样品, 均可有效净化去除杂质, 高效液相色谱图如图1
根据1.3.1免疫磁珠实验方法, 在大米阴性样本中进行低、中、高3水平加标实验。3水平平均回收率为98.2%~106.0%, 相对标准偏差(relative standard deviations, RSDs)为2.56%~4.93%, 如表1所示。根据1.3.2免疫亲和柱法, 在大米阴性样本中进行低、中、高3水平加标实验。3水平加标平均回收率范围为99.4%~102.0%, RSDs为1.03%~2.50%, 如表1所示。比较用两种方法检测大米中AFB1加标结果, 3水平平均回收率均不低于98%, RSDs不超过5.0%。表明大米基质中两者准确度、精密度均较好。
采用两种方法在大米基质中进行低、中、高水平加标实验, 均取得了良好的准确度和精密度。表明免疫亲和柱法和IMB法均适合于大米的检测。大米基质在免疫亲和柱中未引起堵塞; 免疫磁珠在大米基质中充分分散, 抗原抗体充分结合, 洗脱充分, 取得了较好的结果。
向小麦制品阴性基质中添加浓度为2.0 μg/kg的AFB1标准溶液, 分别用两种方法进行检测(n=6), IMB法平均回收率为84.7%, RSD为3.7%; IAC法平均回收率为81.1%, RSD为7.2%。两者平均回收率均在80%~85%之间, 淀粉含量较高, 易引起免疫亲和柱堵塞, 影响回收率结果; 在免疫磁珠实验中, 高淀粉含量可能导致了磁珠聚集, 影响了抗原抗体结合, 从而降低了回收率。但由于其相对更大的表面积, 其精密度表现优于IAC法。
某花生仁炒货中含有AFB1, 采用两种方法对该花生样品中的AFB1含量进行检测, 比较两种方法在检测AFB1含量方面的差异性。t检验结果显示95%置信区间内, 显著性P为0.099, P>0.05。通过t检验结果可知, 在检验油料作物(花生)中AFB1含量时, 两种方法所得结果没有显著性差异。
采用IMB法连续3 d检测加标回收率, 结果见表2。本检测方法连续3 d的平均加标回收率稳定在83.9%~92.2%左右, 日间RSDs在0.51%~1.99%之间, 日间重复性良好。
本研究考察了IMB法, 即免疫磁珠试剂盒联合免疫磁珠纯化仪, 连续3 d测定大米基质加标的回收率和精密度。回收率和精密度均较好。表明该方法稳定可靠, 实验结果具有可信度。
质控样品采用以上两种方法进行测定, 结果如表3。两种方法所得结果均在特性值区间内, 表明两种方法均有较高的准确性。进一步对检验结果分别进行独立样本t检验及Bland-Altman统计分析。
通过对样品进行正态性分布检验和方差齐性检验后, 可知, 质控样品检测结果基本呈正态分布, 且通过了方差齐性检验, 可进行独立样本t检验, 结果显示95%置信区间内显著性P为0.385, P>0.05, 对质控样品的检验两种方法没有显著性差异。
进一步对质控样品采用Bland-Altman方法[30]并统计两者数据是否具有一致性。全部数据分布在控制线内, 两组数据差值的标准偏差(standard deviation, SD)为±1.96SD。说明两种方法检测结果具有较好的一致性, 在检测该基质(玉米粉)中AFB1含量时可以互相代替使用, 结果如图2所示。
以大米、小麦制品(阳春面)、花生制品(花生仁炒货)为基质, 以玉米粉基质质控样品进行质量控制, 采用现行GB 5009.22—2016中第三法 高效液相色谱法 柱后衍生法, 比较了全自动IAC和IMB检测AFB1的效果, 经比较图谱, 两种方法, 净化除杂质方面效果基本相似。免疫磁珠试剂盒效能及免疫亲和柱性能均符合GB 5009.22—2016 附录B.2的要求, 均可进行样品中AFB1含量的检测。
通过稀释法获得大米基质IMB法检出限为0.03 μg/kg, LOQ为0.1 μg/kg, 不低于GB 5009.22—2016 第三法 高效液相色谱法-柱后衍生法(IAC法)LOQ水平。
在大米和小麦制品中进行加标回收率实验, IMB法和IAC法均取得了良好的准确度和精密度。分别采用IMB法和IAC法检测花生AFB1阳性样品, 经独立样本t检验统计, 两种方法统计结果, 没有显著性差异。连续3 d采用IMB法测定大米基质AFB1浓度, 回收率稳定在83%~92%左右, 相对标准偏差在0.5%~2.0%之间。表明方法具有很好的可靠性和稳定性, 这对于确保实验结果的准确性和可信赖性至关重要。
用两种方法对玉米粉基质质控样品进行检测, 结果均在特性值区间内, 说明两种方法准确性均较高。经独立样本t检验和Bland-Altman方法数据统计, 两种方法在检测玉米粉基质中AFB1含量时没有显著性差异, 数据具有一致性, 可以互相代替使用。此外, 全自动免疫磁珠纯化法不仅取得了良好的实验结果, 且实验过程中, 免疫磁珠纯化仪控温准确、温度波动较小; 仪器操作方便、处理简单、比IAC法检测AFB1实验效率高, 一次可同时处理20个样品, 处理量大; 可大量缩短实验时间(净化步骤一次40 min); 产生废液较少(几乎无废液), 绿色环保, 且未有交叉污染情况产生, 同时, 成本可降低约50%; 而免疫亲和柱法净化步骤耗时长, 20批样品耗时约3~4 h; 只能人工操作, 产生大约1000 mL废液。
本研究基本展现出了IMB法检测食品中AFB1的优势, 但其在制备过程中仍有一定的局限性, 导致其在应用过程中遇到易团聚、分散性低等问题。这将是今后磁珠研究过程中重点解决的问题。
  • 面向食品质量安全领域的MOFs高效富集及快速检测关键技术研究项目(22XD1434700)
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2025年第16卷第8期
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doi: 10.19812/j.cnki.jfsq11-5956/ts.20241014004
  • 接收时间:2024-10-14
  • 首发时间:2025-07-19
  • 出版时间:2025-04-25
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  • 收稿日期:2024-10-14
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面向食品质量安全领域的MOFs高效富集及快速检测关键技术研究项目(22XD1434700)
作者信息
    1.上海市松江食品药品检验所, 上海 201306
    2.月旭科技(上海)股份有限公司, 上海 201613
    3.上海理工大学, 上海食品快速检测工程技术研究中心, 上海 200093

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* 石春红(1969—), 女, 硕士, 正高级工程师, 主要研究方向为食品检验质量控制及管理。E-mail:
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2种不同金属材料的力学参数

Family
属数
Number of
genus
种数
Number of
species
占总种数比例
Percentage of
total species (%)

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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