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黄曲霉毒素B1高灵敏定性定量免疫层析检测方法的建立

章先 王继璇 程高钏 李可 张晓峰 孙孟娇 程昌勇 宋厚辉

章先, 王继璇, 程高钏, 李可, 张晓峰, 孙孟娇, 程昌勇, 宋厚辉. 黄曲霉毒素B1高灵敏定性定量免疫层析检测方法的建立[J]. 浙江农林大学学报. doi: 10.11833/j.issn.2095-0756.20210772
引用本文: 章先, 王继璇, 程高钏, 李可, 张晓峰, 孙孟娇, 程昌勇, 宋厚辉. 黄曲霉毒素B1高灵敏定性定量免疫层析检测方法的建立[J]. 浙江农林大学学报. doi: 10.11833/j.issn.2095-0756.20210772
ZHANG Xian, WANG Jixuan, CHENG Gaochuan, LI Ke, ZHANG Xiaofeng, SUN Mengjiao, CHENG Changyong, SONG Houhui. Development of a highly sensitive qualitative and quantitative immunochromatographic method for the detection of aflatoxin B1[J]. Journal of Zhejiang A&F University. doi: 10.11833/j.issn.2095-0756.20210772
Citation: ZHANG Xian, WANG Jixuan, CHENG Gaochuan, LI Ke, ZHANG Xiaofeng, SUN Mengjiao, CHENG Changyong, SONG Houhui. Development of a highly sensitive qualitative and quantitative immunochromatographic method for the detection of aflatoxin B1[J]. Journal of Zhejiang A&F University. doi: 10.11833/j.issn.2095-0756.20210772

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黄曲霉毒素B1高灵敏定性定量免疫层析检测方法的建立

doi: 10.11833/j.issn.2095-0756.20210772
基金项目: 浙江省重点研发计划项目(2021C02058);国家自然科学基金资助项目(32002358);国家级大学生创新创业训练计划项目(202010341040)
详细信息
    作者简介: 章先(ORCID: 0000-0003-4903-3009),讲师,博士,从事病原生物学研究。E-mail: zhangxian073@163.com
    通信作者: 宋厚辉(ORCID: 0000-0001-6530-5794),教授,博士,从事病原生物学研究。E-mail: songhh@zafu.edu.cn
  • 中图分类号: S859.84

Development of a highly sensitive qualitative and quantitative immunochromatographic method for the detection of aflatoxin B1

  • 摘要:   目的  真菌毒素可污染农产品和动物源性食品,其中黄曲霉毒素B1(AFB1)毒性强、危害大,建立AFB1快速、高灵敏和便捷的检测方法对于监测相关产品中AFB1污染水平,保障人和动物健康均具有重要意义。本研究基于侧向层析技术原理,采用竞争模式,优化建立免疫层析检测方法,以实现AFB1的快速定性检测和定量分析。  方法  通过比较分析不同粒径金颗粒标记抗体效果,优化确定免疫层析各组分材料类型、相关缓冲液配方及最佳使用质量浓度,建立AFB1高灵敏定性定量免疫层析检测方法。  结果  优化建立的AFB1免疫层析检测法在实际样本中的定性和定量检测限分别为2.5和0.5 μg·kg−1,灵敏度高、特异性强,与其他常见真菌毒素无交叉反应,加标回收实验结果显示:该方法准确稳定,且对AFB1天然污染样本的定量检测结果与商品化试剂盒及LC-MS/MS一致性较好。  结论  本研究制备的免疫层析检测法可用于样本中AFB1污染的快速定性检测与定量分析,适合缺乏实验条件的基层检验检疫机构和农产品加工企业对大量样本进行快速筛查,样本检测结果疑似阳性再采用仪器法进行确认,可降低检测成本,提升检测效率,同时为建立其他病原微生物免疫层析检测方法提供参考。图9表2参26
  • 图  1  黄曲霉毒素B1定性定量免疫层析检测法示意图

    Figure  1  Schematic diagram of the qualitative and quantitative immunochromatographic assay for the detection of aflatoxin B1

    图  2  免疫层析体系各组分组成结构示意图

    Figure  2  Sketch map of the immunochromatographic system

    图  3  AFB1完全抗原AFB1-BSA和AFB1-OVA的ELISA鉴定

    Figure  3  Absorbance of the AFB1 conjugates by indirect ELISA using their specific monoclonal antibodies

    图  4  目测法(A)和透射电镜(B)对金颗粒的鉴定

    Figure  4  Colloidal solution of 20 nm and 40 nm diameter gold nanoparticles by visualization by naked eye(A) and images from TEM(B)

    图  5  免疫层析法对黄曲霉毒素B1的定性检测限

    Figure  5  Detection limits of immunochromatographic assay for aflatoxin B1

    图  6  免疫层析法对黄曲霉毒素B1的抑制曲线(A)和定量标准曲线(B)

    Figure  6  Tri-parametric curve fitting of log concentration of aflatoxin B1 vs inhibition rate by immunochromatographic assay(A) and the calibration curve for quantification of aflatoxin B1(B)

    图  7  定性定量免疫层析法对其他真菌毒素的交叉反应性

    Figure  7  Cross-reactivities of the qualitative and quantitative immunochromatographic assay for different mycotoxins

    图  8  免疫层析法对黄曲霉毒素B1加标样本的定性检测

    Figure  8  Qualitative detection of aflatoxin B1 spiked samples by immunochromatography

    图  9  免疫层析法与LC-MS/MS定量结果相关性分析

    Figure  9  Correlation of results obtained by immunochromatography assay and LC-MS/MS for AFB1 detection in natural samples

    表  1  免疫层析法对黄曲霉毒素B1加标样本的定量检测

    Table  1.   Quantitative detection of aflatoxin B1 spiked samples by immunochromatography assay

    AFB1加标质量浓
    度/(μg·kg−1)
    批内批间
    回收
    率/%
    变异系
    数/%
    回收
    率/%
    变异系
    数/%
    1.089.62 ± 5.315.9395.72 ± 8.038.39
    2.596.47 ± 6.356.58103.56 ± 7.547.28
    5.095.16 ± 4.294.5193.25 ± 9.069.72
    10.0110.43 ± 6.155.57107.59 ± 8.567.96
      说明:数据为平均值±标准差,n=3
    下载: 导出CSV

    表  2  免疫层析法、商品化试剂盒和LC/MS/MS对天然样本中黄曲霉毒素B1的定量检测结果

    Table  2.   Quantitative detection of AFB1 in natural samples by the developed immunochromatography assay, commercial ELISA kit and LC-MS/MS

    样本编号AFB1/(μg·kg−1)样本编号AFB1/(μg·kg−1)
    免疫层析检测法LC/MS/MS商品化试剂免疫层析检测法LC/MS/MS商品化试剂
    15.11 ± 0.324.79 ± 0.295.65 ± 0.3781.83 ± 0.142.15 ± 0.272.71 ± 0.19
    23.17 ± 0.252.52 ± 0.162.08 ± 0.1892.09 ± 0.172.86 ± 0.253.57 ± 0.26
    31.06 ± 0.111.34 ± 0.142.33 ± 0.32108.65 ± 0.357.21 ± 0.628.94 ± 0.19
    46.63 ± 0.475.83 ± 0.435.36 ± 0.57114.63 ± 0.295.93 ± 0.384.38 ± 0.23
    52.69 ± 0.223.15 ± 0.292.25 ± 0.26125.27 ± 0.416.07 ± 0.575.66 ± 0.35
    61.82 ± 0.132.57 ± 0.153.35 ± 0.18133.28 ± 0.372.19 ± 0.183.95 ± 0.31
    77.16 ± 0.516.03 ± 0.317.82 ± 0.23
      说明:数据为平均值±标准差
    下载: 导出CSV
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  • 收稿日期:  2021-11-29
  • 录用日期:  2022-04-01
  • 修回日期:  2022-03-31

黄曲霉毒素B1高灵敏定性定量免疫层析检测方法的建立

doi: 10.11833/j.issn.2095-0756.20210772
    基金项目:  浙江省重点研发计划项目(2021C02058);国家自然科学基金资助项目(32002358);国家级大学生创新创业训练计划项目(202010341040)
    作者简介:

    章先(ORCID: 0000-0003-4903-3009),讲师,博士,从事病原生物学研究。E-mail: zhangxian073@163.com

    通信作者: 宋厚辉(ORCID: 0000-0001-6530-5794),教授,博士,从事病原生物学研究。E-mail: songhh@zafu.edu.cn
  • 中图分类号: S859.84

摘要:   目的  真菌毒素可污染农产品和动物源性食品,其中黄曲霉毒素B1(AFB1)毒性强、危害大,建立AFB1快速、高灵敏和便捷的检测方法对于监测相关产品中AFB1污染水平,保障人和动物健康均具有重要意义。本研究基于侧向层析技术原理,采用竞争模式,优化建立免疫层析检测方法,以实现AFB1的快速定性检测和定量分析。  方法  通过比较分析不同粒径金颗粒标记抗体效果,优化确定免疫层析各组分材料类型、相关缓冲液配方及最佳使用质量浓度,建立AFB1高灵敏定性定量免疫层析检测方法。  结果  优化建立的AFB1免疫层析检测法在实际样本中的定性和定量检测限分别为2.5和0.5 μg·kg−1,灵敏度高、特异性强,与其他常见真菌毒素无交叉反应,加标回收实验结果显示:该方法准确稳定,且对AFB1天然污染样本的定量检测结果与商品化试剂盒及LC-MS/MS一致性较好。  结论  本研究制备的免疫层析检测法可用于样本中AFB1污染的快速定性检测与定量分析,适合缺乏实验条件的基层检验检疫机构和农产品加工企业对大量样本进行快速筛查,样本检测结果疑似阳性再采用仪器法进行确认,可降低检测成本,提升检测效率,同时为建立其他病原微生物免疫层析检测方法提供参考。图9表2参26

English Abstract

章先, 王继璇, 程高钏, 李可, 张晓峰, 孙孟娇, 程昌勇, 宋厚辉. 黄曲霉毒素B1高灵敏定性定量免疫层析检测方法的建立[J]. 浙江农林大学学报. doi: 10.11833/j.issn.2095-0756.20210772
引用本文: 章先, 王继璇, 程高钏, 李可, 张晓峰, 孙孟娇, 程昌勇, 宋厚辉. 黄曲霉毒素B1高灵敏定性定量免疫层析检测方法的建立[J]. 浙江农林大学学报. doi: 10.11833/j.issn.2095-0756.20210772
ZHANG Xian, WANG Jixuan, CHENG Gaochuan, LI Ke, ZHANG Xiaofeng, SUN Mengjiao, CHENG Changyong, SONG Houhui. Development of a highly sensitive qualitative and quantitative immunochromatographic method for the detection of aflatoxin B1[J]. Journal of Zhejiang A&F University. doi: 10.11833/j.issn.2095-0756.20210772
Citation: ZHANG Xian, WANG Jixuan, CHENG Gaochuan, LI Ke, ZHANG Xiaofeng, SUN Mengjiao, CHENG Changyong, SONG Houhui. Development of a highly sensitive qualitative and quantitative immunochromatographic method for the detection of aflatoxin B1[J]. Journal of Zhejiang A&F University. doi: 10.11833/j.issn.2095-0756.20210772

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