基于适配体识别的金黄色葡萄球菌定性检测试纸条的研制及应用

本研究以金黄色葡萄球菌为检测靶标，以核酸适配体为识别分子，基于双适配体夹心和侧流层析原理，构建了定性检测金黄色葡萄球菌的适配体试纸条，并对NaCl浓度、适配体浓度、纳米金-适配体包被量及捕获探针包被量等实验条件进行优化，获得适配体试纸条最佳制备条件。在优化条件下对试纸条的灵敏度、特异性进行分析测试，最后将试纸条对116份食品进行金黄色葡萄球菌检测，并与国标法（GB 4789.10-2016）对比验证。结果显示，适配体试纸条最佳制备条件为NaCl浓度为80 mmol/L、适配体偶联浓度为1 μmol/L、结合垫上纳米金-适配体的稀释体积比为1:2、捕获探针浓度为25 μmol/L。在最佳条件下，适配体试纸条对金黄色葡萄球菌的可视化检测限为2×103 CFU/mL，检测时间为5 min，且与其他食源性致病菌如鼠伤寒沙门氏菌、单增李斯特菌、大肠杆菌、阪崎肠杆菌、痢疾志贺氏菌等无交叉反应，具有较高的特异性。将本方法应用于食品中金黄色葡萄球菌的定性检测，检测结果与国标法完全一致。该方法简便快速、准确可靠、成本低，适用于食品样品中金黄色葡萄球菌的定性检测。

Study of Aptamer-Based Lateral Flow Strip and Its Application for Qualitative Detection of Staphylococcus aureus

In this experiment, Staphylococcus aureus was selected as the model target, and a pair of aptamers were used as the recognition molecules. An aptamer-based lateral flow test strip (ABLF) was prepared for the qualitative detection of Staphylococcus aureus based on the sandwich-type model and principle of lateral flow chromato gram. Several crucial parameters were investigated, including the NaCl concentrations, aptamer concentrations, the loaded amounts of gold nanoparticle-aptamer conjugates and capture probe to obtain the best preparation conditions for ABLF. The sensitivity and specificity of the ABLF were tested under optimized conditions. Finally, the ABLF approach was applied for qualitative detection of Staphylococcus aureu in 116 food samples, and was validated using sandard method (GB 4789.10-2016). The results showed that the optimal conditions for preparing ABLF were as follows: NaCl concentration 80 mmol/L, aptamer concentration 1 μmol/L, dilution volume ratio of gold nanoparticles-aptamer 1:2, capture probe concentration 25 μmol/L. Under the optimal experimental conditions, the visual limit of detection of the strip for Staphylococcus aureus were 2×103 CFU/mL. The assay can be completed within 5 min, and no significant cross-reactivity with other foodborne pathogens such as Salmonella typhimurium, Listeria monocytogenes, Escherichia coli, Bntorobater sakazakii, Shigella dysenteriae were observed. The test results obtained by the proposed method were found to be in consistency with those obtained from GB sandard method. The method developed was simple, rapid, accurate and inexpensive, which could be a potential screening tool for the qualitative detection of Staphylococcus aureus in food samples.