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。
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.
作者
林祥群
杨国江
卢春霞
陶思桦
闫圣坤
LIN Xiangqun;YANG Guojiang;LU Chunxia;TAO Sihua;YAN Shengkun(Xinjiang Shihezi Vocational Technical College Textile and Chemical Branch,Shihezi 832000,China;Institute of Irrigation and Soil Fertilizer,Xinjiang Academy of Agriculture and Reclamation Science,Shihezi 832000,China;School of Advanced Agriculture and Bioengineering,Yangtze Normal University,Chongqing 408100,China;Institute of Agricultural Mechanization Xinjiang Academy of Agricultural Sciences,Urumqi 830091,China)
出处
《现代食品科技》
CAS
北大核心
2022年第2期305-312,共8页
Modern Food Science and Technology
基金
重庆市自然科学基金面上项目(cstc2020jcyj-msxmX0080)
苏州相城区科技领军人才计划。
