基于核酸适配体的AccuBlue荧光法检测动物食品中的恩诺沙星

A Fluorescence Method Based on AccuBlue and Nucleic Acid Aptamer for Detection of Enrofloxacin in Animal-Derived Foods

AccuBlue荧光染料在游离或与单链DNA共存时,只显示微弱的荧光,与双链DNA共存时,其荧光信号显著增强,利用此特点,建立一种基于核酸适配体的恩诺沙星荧光检测方法。最优检测条件为在pH 8.5的缓冲溶液中,双链最佳孵育时间10 min,荧光染料识别双链的时间6 min。结果表明,该方法在检测范围为20~1000μg/L内线性关系良好,检出限为9.96μg/L,定量限为29.97μg/kg。板内变异系数范围为4.97%~9.31%,板间变异系数范围为5.83%~10.96%。同时,选取3种动物源性食品(奶粉、虾肉和牛肉)进行加标回收实验,在3个不同水平,加标回收率在76.54%~98.79%之间。说明所构建AccuBlue荧光法的特异性、稳定性和重复性均良好,可用于实际样品的快速检测。

AccuBlue fluorescent dye displays only weak fluorescence when being free or coexisting with single-stranded DNA,but significantly enhanced fluorescent signals when coexisting with double-stranded DNA.In this study,a novel fluorescence method for enrofloxacin detection was established based on the reaction between AccuBlue fluorescent dye and a nucleic acid aptamer.The optimal detection conditions were as follows:in a buffer solution at pH 8.5,the double strand was incubated for 10 min,and the fluorescent dye was allowed 6 min to recognize the double strand.Results indicated that the calibration curve was linear in the concentration range of 20–1000μg/L.Validation of the method yielded a limit of detection(LOD)(RSN≥3)of 9.96μg/L and a limit of quantification(LOQ)(RSN≥10)of 29.97μg/kg.The intra-and inter-plate variation coefficients were in the ranges of 4.97%–9.31%and 5.83%–10.96%,respectively.Recoveries measured for the extraction of enrofloxacin spiked into milk powder,shrimp and beef were between 76.54%and 98.79%.The method developed proved to be specific,stable,and repeatable,and could be easily implemented for rapid detection of enrofloxacin in real samples.