卡那霉素A酶联适配体检测方法研究

Development of Enzyme-linked Aptamer Assay for Detection of Kanamycin A

通过EDC法偶联辣根过氧化物酶(HRP)和卡那霉素A(Kanamycin,KaA)分子制备酶标记物,采用棋盘滴定法确定链霉亲和素(Streptavidin,SA)的包被浓度为8 mg/L,通过单因素实验优化了检测条件,建立了卡那霉素A酶联适配体检测(Enzyme-linked aptamer assay,ELAA)方法。本方法半抑制浓度(IC50)为2.2"g/L,检出限(LOD)为0.04"g/L,检测范围(IC20～IC80)为0.07～71.5"g/L,与结构类似物无明显交叉反应;对牛奶、猪肉、猪肝、鸡肉、蜂蜜样品加标回收率在78%～100%之间,平均相对标准偏差小于11.1%。本方法特异性强、灵敏度高,适用于食品中卡那霉素A的快速检测。

The EDC method was employed to conjugate the kanamycin A（KaA） to horseradish peroxidase（HRP）.The optimal coating concentration of streptavidin was 8 mg/L and that was determined by chequerboard titration.The optimized assay conditions were investigated by single-factor experiments.An enzyme-linked aptamer assay（ELAA） method was established for the determination of kanamycin A（KaA）.The half inhibition concentration（IC50） of the proposed method was 2.2 μg/L,the limit of detection（LOD） was 0.04 μg/L,and the detection ranged from 0.07 μg/L to 71.5 μg/L,There were no conspicuous cross reactions with other structural analogues of KaA.The recovery in milk,pork,pork liver,chicken and honey samples was 78%-100% and the RSD was less than 11.1%.This method is specific and highly sensitive,suitable for the rapid detection of kaA in variable food samples.