黄曲霉毒素B_(1)高灵敏定性定量免疫层析检测方法的建立  

A highly sensitive qualitative and quantitative immunochromatographic method for the detection of aflatoxin B_(1)

作　　者：章先 王继璇[1] 程高钏 李可 张晓峰 孙孟娇[4] 程昌勇 宋厚辉 ZHANG Xian;WANG Jixuan;CHENG Gaochuan;LI Ke;ZHANG Xiaofeng;SUN Mengjiao;CHENG Changyong;SONG Houhui(College of Medicine,Zhejiang University,Hangzhou 310058,Zhejiang,China;College of Animal Science and Technology/College of Veterinary Medicine,Zhejiang A&F University,Hangzhou 311300,Zhejiang,China;Zhejiang Academy of Science and Technology for Inspection and Quarantine,Hangzhou 311202,Zhejiang,China;College of Animal Science,Zhejiang University,Hangzhou 310058,Zhejiang,China)

机构地区：[1]浙江大学医学院,浙江杭州310058 [2]浙江农林大学动物科技学院/动物医学院,浙江杭州311300 [3]浙江省检验检疫科学技术研究院,浙江杭州311202 [4]浙江大学动物科技学院,浙江杭州310058

出　　处：《浙江农林大学学报》2022年第5期1096-1103,共8页Journal of Zhejiang A&F University

基　　金：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)。

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B_(1)(AFB_(1))毒性强、危害大,建立AFB_(1)快速、高灵敏和便捷的检测方法对于监测相关产品中AFB污染水平,保障人和动物健康均具有重要意义。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AFB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AFB高灵敏定性定量免疫层析检测方法。【结果】优化建立的AFB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0.5μg·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AFB_(1)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AFB_(1)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。[Objective] Fungal metabolites, commonly known as mycotoxins, can pollute agricultural products and food of animal origin, among which aflatoxin B_(1)(AFB_(1)) is the most common, toxic and detrimental.Establishing a rapid, highly sensitive and convenient detection method of AFB_(1)is of great significance for the protection of human and animal health. The objective of this study is to optimize the immunochromatographic detection method based on the principle of lateral-flow chromatography and competitive mode, so as to realize the rapid qualitative detection and quantitative analysis of AFB_(1). [Method] A highly sensitive qualitative and quantitative immunochromatographic detection method for AFB_(1)was established by comparing and analyzing the labeling effects of gold particles of varying sizes,optimizing the material types of each component of immunochromatography,as well as relevant buffer solution and the optimal mass concentration.[Result]The qualitative and quantitative detection limits of the optimized AFB_(1)immunochromatographic method in samples were 2.5 and 0.5μg·kg^(-1),respectively,with high sensitivity and specificity and no cross reaction with other common mycotoxins.Standard addition recovery experiment showed that the method was accurate and stable,and the quantitative detection results of AFB_(1)natural contamination samples were in good agreement with commercial kit and LC-MS/MS.[Conclusion]The immunochromatographic detection method prepared in this study can be used for rapid qualitative detection and quantitative analysis of AFBcontamination in samples.It is suitable for grass-roots inspection and quarantine institutions and agricultural product processing enterprises that lack experimental conditions to quickly screen a large number of samples.If the sample test result is suspected to be positive,the instrument method can be used for confirmation,which can reduce the test cost,improve the test efficiency and provide reference for the establishment of immunochromatographic detection meth

关 键 词：定性检测 定量分析 黄曲霉毒素B_(1) 免疫层析 单克隆抗体 

分 类 号：S859.84[农业科学—临床兽医学]