基于酶联适配体快速检测食品中葡萄球菌肠毒素A

Colorimetric Enzyme-linked Aptamer Assay for Determination of Staphylococcal Enterotoxin A in Foods

摘要:

该研究基于双适配体夹心原理，建立了一种酶联适配体检测葡萄球菌肠毒素A（Staphylococcal enterotoxin A，SEA）的新方法，并对适配体浓度、链霉亲和素-辣根过氧化物酶浓度、反应体系、封闭条件、反应时间等条件进行了优化。结果显示，在适配体浓度40 nmol/L、牛血清白蛋白（Bovine serum albumin，BSA）封闭浓度为1%、封闭时间4 h、反应体系为SEA适配体筛选缓冲液、链霉亲和素-辣根过氧化物酶稀释体积比1:40000、靶标与适配体反应时间30 min等优化条件下，SEA在5~500 ng/mL浓度范围内呈良好线性（R=0.996），方法检出限为0.18 ng/mL，加标回收率为91.22%~101.30%，批内和批间相对标准偏差为2.82%~7.50%。将建立的方法应用于食品样品中SEA检测，并与国标法（GB 4789.10-2016）对比验证，两种方法的检测结果具有高的相关性（R=0.994）。以上结果表明本方法具有高的灵敏度、准确性和特异性性，该研究为食品中SEA检测提供一种简便、经济、高通量的快速检测新技术。

Abstract:

In this study, an enzyme-linked aptamer assay (ELAA) was developed for detection of staphylococcus enterotoxin A (SEA) based on sandwich-type format strategy. Several experimental conditions have been investigated, including the concentration of aptamer, BSA and streptavidin-horseradish peroxidase (SA-HRP), reactionsystem, blocking conditions, and incubation time between SEA and aptamer. The results showed that the optimal conditions for ELAA were as follows: aptamers concentration of 40 nmol/L; BSA concentration of 1%; blocking time of 4 h; SEA aptamer screening buffer was used as the reaction system; the dilution volume ratio of streptavidin-horseradish peroxidase (SA-HRP) was 1:40000; and the incubation time between SEA and aptamers was 30 min. Under the optimal conditions, the calibration curves for SEA showed good linearity in the range of 5~500 ng/mL with correlation coefficients (R) better than 0.996. The limit of detection (LOD) for SEA was 0.18 ng/mL. The recoveries of SEA in food samples were in the range of 91.22%~101.30%. There lative standard deviation (RSD) of intra-assay and inter-assay were in the range of 2.82%~7.50%. Subsequently, the proposed method was applied to measure SEA in real samples, and was validated using official standard method. A good correlation (R=0.994) was obtained between the results of the two methods. These results suggest that the proposed method had good sensitivity, accuracy and specificity. This study provides a simple, economical and high-throughput new rapid detection technology for SEA detection in food.