基于核酸适配体免标记光度法检测牛奶中的甲硝唑

Determination of metronidazole in milk based on nucleic acid aptamer labeling free spectrophotometry

目的建立核酸适配体免标记光度法检测牛奶中的甲硝唑的分析方法。方法将含DNA酶序列的核酸探针与甲硝唑适配体混合反应,引入目标检测物甲硝唑,使其与甲硝唑适体特异性结合,再于体系中引入氧化还原试剂,用紫外可见分光光度计检测溶液吸光度变化。优化各反应条件,以达到最佳反应条件,最后对所构建的方法进行方法学评价。结果当甲硝唑质量浓度在0.08~5.13 g/L时,体系的荧光强度差值(ΔA)与甲硝唑质量浓度(C)呈线性关系,ΔA=0.1542+0.1396C(r=0.9968),检出限为0.0358 g/L。在低、中、高3水平浓度的加标回收率为85.91%~102.04%,相对标准偏差分别为1.95%、1.93%、1.36%。结论本方法具有良好的精密度、选择性、回收率,可用于实际样品牛奶中甲硝唑的检测。

Objective To establish a method for the determination of metronidazole in milk by nucleic acid aptamer labeling free spectrophotometry.Methods The nucleic acid probe containing DNA enzyme sequence was mixed with the aptamer of metronidazole,and the target detection object metronidazole was introduced to make it specifically bind with the aptamer of metronidazole.Then the redox reagent was introduced into the system,and the change of absorbance of the solution was detected by ultraviolet visible spectrophotometer.Various conditions were optimize to achieve the best reaction conditions.Finally,methodological evaluation was conducted for the constructed methods.Results The difference in fluorescence intensity(ΔA)of the system showed a linear relationship with the metronidazole mass concentration(C)when the mass concentration of chloramphenicol were in the range of 0.08‒5.13 g/L,and the linear equation wasΔA=0.1542+0.1396C(r=0.9968).The limit of detection for the method were calculated as 0.0358 g/L.The recoveries of low,medium and high levels were 85.91%‒102.04%with relative standard deviations of 1.95%,1.93%and 1.36%,respectively.Conclusion The method has good precision,selectivity and recovery,and can be used for the determination of metronidazole in milk.