农产品中黄曲霉毒素的时间分辨荧光免疫层析快速检测技术研究

doi:10.3864/j.issn.0578-1752.2014.18.014

中国农业科学 ›› 2014, Vol. 47 ›› Issue (18): 3668-3674.doi: 10.3864/j.issn.0578-1752.2014.18.014

农产品中黄曲霉毒素的时间分辨荧光免疫层析快速检测技术研究

张兆威，李培武，张奇，丁小霞

中国农业科学院油料作物研究所/农业部生物毒素检测重点实验室/农业部油料作物生物学与遗传育种重点实验室/农业部油料产品质量安全风险评估实验室（武汉）/农业部油料及制品质量监督检验测试中心，武汉 430062

收稿日期:2014-03-25 修回日期:2014-05-27 出版日期:2014-09-16 发布日期:2014-09-16
通讯作者: 李培武，Tel：027-86812943；E-mail：peiwuli@oilcrops.cn
作者简介:张兆威，Tel：027-86711839；E-mail：zwzhang@whu.edu.cn
基金资助:
国家科技支撑计划（2012BAB19B09）
国家公益性行业（农业）科研专项（201203094）
国家自然科学基金（31101299、31171702）

Study on Immunochromatography for Afaltoxin Determination in Agricultural Product

ZHANG Zhao-wei, LI Pei-wu, ZHANG Qi, DING Xiao-xia

Oil Crops Research Institute, Chinese Academy of Agricultural Sciences/Key Laboratory of Detection for Mycotoxins, Ministry of Agriculture/Key Laboratory of Biology and Genetic Improvement of Oil Crops, Ministry of Agriculture/Laboratory of Risk Assessment for Oilseeds Products (Wuhan), Ministry of Agriculture/Quality Inspection and Test Center for Oilseeds Products, Ministry of Agriculture, Wuhan 430062

Received:2014-03-25 Revised:2014-05-27 Online:2014-09-16 Published:2014-09-16

摘要/Abstract

摘要： 【目的】黄曲霉毒素严重污染农产品，威胁人畜生命健康，是政府重视、社会关注的一大焦点问题，因此亟需研究建立黄曲霉毒素高灵敏高快速检测方法。目前已研制出高特异性高灵敏度黄曲霉毒素单克隆抗体，本研究旨在应用该抗体，建立黄曲霉毒素时间分辨荧光免疫层析快速检测技术，为农产品质量监管、风险评估提供技术支持。【方法】本研究通过将黄曲霉毒素抗体与乳胶铕进行偶联标记，利用自主研制的黄曲霉毒素时间分辨荧光免疫层析试纸条进行黄曲霉毒素检测，通过检测线T信号值与质控线C信号值的比值和标准溶液浓度的自然对数值实现定量。针对花生、稻米、植物油等不同农产品，研究建立样品粉碎均质提取一体化技术。对铕标记时间分辨荧光免疫层析检测技术进行方法学考核，以花生、稻米、植物油等为例，进行实际样品检测，并与HPLC法进行结果比对。【结果】在花生、稻米、植物油等农产品实际样品检测中，本研究建立的时间分辨荧光免疫层析检测技术检测黄曲霉毒素B₁得到检测限均为0.3 μg·kg^-1，线性范围分别为0.8—25、0.8—15和0.8—30 μg·kg^-1。上述3种农产品对应的标准曲线的线性方程分别为y=0.238x+0.654（R²=0.992）、y=0.321x+0.811（R²=0.990）和y=0.146x+0.173（R ²=0.993），标准偏差分别为0.024、0.039和0.021。该方法的批内准确度和精密度添加回收试验结果表明，添加回收率在81.0%—113.0%，变异系数在7.2%—14.2%；批间准确度和精密度试验结果显示，添加回收率在75.8%—114.9%，变异系数在7.7%—15.3%。说明该检测技术批内、批间均具有良好的准确度和精密度。该方法与HPLC法检测结果相比，相对误差小于10%，说明黄曲霉毒素时间分辨荧光免疫层析技术与行业标准方法检测结果具有良好的一致性。【结论】时间分辨荧光免疫层析检测技术灵敏度高、线性范围宽，重复性和稳定性好，是一种适合中国国情的实用快速检测技术，具有广阔的应用前景。

关键词: 黄曲霉毒素, 时间分辨荧光免疫层析, 农产品, 快速检测

Abstract: 【Objective】 Aflatoxin contaminates agricultural product severely, threatens health and life of people and livestocks. It is one of the major focus issues and attracts both governmental and social concerns. Thus, it is required to establish a rapid and sensitive determination method for aflatoxin. High specific and sensitive monoclonal antibody against aflatoxin has been developed. In this study, using the as-prepared monoclonal antibody against aflatoxin, the aim is to establish time-resolved fluorescence immunochromatography for afaltoxin determination in agricultural product, in order to provide technical support for agricultural product quality supervision and risk assessment. 【Method】 Herein, monoclonal antibody against aflatoxin was coupled with emulsion europium for labeling. With the home-made time-resolved fluorescence immunochromatographic strip, aflatoxin could be determined quantitatively, by using the signal value ratio of test line to control line and natural logarithm of concentration in standard aflatoxin solution. Regarding to various agricultural product samples (as peanut, rice and vegetable oil), an integration technology was developed that combined grinding and homogenization in one step. The methodological evaluation was conducted, in which aflatoxin in real agricultural products such as peanut, rice and vegetable oil. Moreover, these results via time-resolved fluorescence immunochromatography were compared with those via HPLC method. 【Result】 Results showed a detecting limit of 0.3 μg·kg^-1, a liner range of 0.8-25, 0.8-15, 0.8-30 μg·kg^-1, for peanut, rice, plant oil, respectively. The standard curves were recorded as y=0.238x+0.654 (R²=0.992), y=0.321x+0.811 (R²=0.990), and y=0.146x+0.173 (R²=0.993), for peanut, rice, and plant oil, respectively. Recoveries from inner accuracy and precision were between 81.0% and 113.0% with RSD of 7.2%-14.2%, while recoveries from inter accuracy and precision were of 75.8%-114.9% with RSD of 7.7%-15.3%. It was suggested that this method allowed considerable inner- and inter- accuracy and precision, It was found to be the relative error below 10% between results via time-resolved fluorescence immunochromatography and those with HPLC, which suggesting that a high degree between time-resolved fluorescence immunochromatographic method and industry standard method.【Conclusion】Results showed that TRFIA possesses high sensitivity, wide liner range, high repeatability and stability, thus it is suitable for rapid assay in China and poses wide application prospect.

Key words: aflatoxin, time-resolved fluorescence immunochromatography, agricultural product, rapid assay

张兆威，李培武，张奇，丁小霞. 农产品中黄曲霉毒素的时间分辨荧光免疫层析快速检测技术研究[J]. 中国农业科学, 2014, 47(18): 3668-3674.

ZHANG Zhao-wei, LI Pei-wu, ZHANG Qi, DING Xiao-xia. Study on Immunochromatography for Afaltoxin Determination in Agricultural Product[J]. Scientia Agricultura Sinica, 2014, 47(18): 3668-3674.

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农产品中黄曲霉毒素的时间分辨荧光免疫层析快速检测技术研究

Study on Immunochromatography for Afaltoxin Determination in Agricultural Product

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