基于核酸适配体的金黄色葡萄球菌快速检测试纸条的研制和应用

doi:10.16431/j.cnki.1671-7236.2023.11.026

摘要/Abstract

摘要： 【目的】建立能快速检测金黄色葡萄球菌的胶体金免疫层析法。【方法】制备胶体金并表征，将其与核酸适配体连接后进行表征并喷涂于结合垫，以金黄色葡萄球菌多克隆抗体为检测线、核酸适配体互补序列为质控线制备试纸条，对样品垫与结合垫处理液、样品稀释液、重悬液、T线抗体浓度、适配体连接浓度等试验条件进行优化以获得最佳条件，同时检测试纸条的增敏效果，在最佳条件下对试纸条的灵敏度、特异性进行测试，制备人工污染样品用以评估试纸条在实际样品检测中的效果。【结果】胶体金粒径为20 nm左右，表征结果显示，胶体金与核酸适配体成功连接；试纸条样品垫与结合垫处理液、样品稀释液的最优条件为不同成分的SELEX缓冲液，金标适配体重悬液为HEPES重悬液，T线抗体浓度为2 mg/mL，适配体连接浓度为1 μmol/L；用40 mmol/L羟胺与0.5%氯化金水溶液增敏，检测灵敏度为1×10⁴ CFU/mL，且具有良好的特异性，对于人工污染的肉样和奶样进行检测，灵敏度为1×10⁵ CFU/mL。【结论】本研究成功建立金黄色葡萄球菌试纸条检测方法，且该方法特异性良好，为进一步探讨核酸适配体在病原菌检测中的应用提供了理论依据。

关键词: 核酸适配体; 胶体金; 金黄色葡萄球菌; 快速检测

Abstract: 【Objective】 The aim of this experiment was to construct a colloidal gold immunochromatography for the rapid detection of Staphylococcus aureus.【Method】 The colloid gold was prepared and characterized, and then connected with aptamer, characterized and sprayed on the binding pad.The test strip was constructed with polyclonal antibody as the test line and the complementary sequence of aptamer as the control line.The test conditions of sample pad and binding pad treatment solution, sample dilution solution, suspension solution, T-line antibody concentration, and aptamer connection concentration were optimized to obtain the best conditions.At the same time, the sensitization effect of the test strip was studied, and the sensitivity and specificity of the test strip were tested under the best conditions.Finally, the artificial contaminated sample was prepared to evaluate the effect of the test strip in the actual sample detection.【Result】 The particle size of colloidal gold was about 20 nm, and the results showed that the colloidal gold was successfully connected to the aptamer.The optimal conditions for the sample pad, the binding pad and the sample diluent were SELEX buffer with different components, the resuspension with gold standard was HEPES resuspension, and the T-line antibody concentration was 2 mg/mL.The aptamer binding concentration was 1 μmol/L, and 40 mmol/L hydroxylamine was sensitized with 0.5% gold chloride aqueous solution, and the detection sensitivity was 1×10⁴ CFU/mL with good specificity.For artificially contaminated meat and milk samples, the sensitivity was 1×10⁵ CFU/mL.【Conclusion】 The test strip detection method for Staphylococcus aureus was established successfully, and had good specificity, which provided a theoretical basis for further exploring the application of nucleic acid aptamer in the detection of pathogenic bacteria.

Key words: aptamers; colloidal gold; Staphylococcus aureus; rapid detection

中图分类号:

S852.61

李磊, 肖爽, 甄思慧, 王瑜, 武周慧, 王之文, 杜衡, 鲁琳, 王真. 基于核酸适配体的金黄色葡萄球菌快速检测试纸条的研制和应用[J]. 中国畜牧兽医, 2023, 50(11): 4577-4588.

LI Lei, XIAO Shuang, ZHEN Sihui, WANG Yu, WU Zhouhui, WANG Zhiwen, DU Heng, LU Lin, WANG Zhen. Development and Application of Nucleic Acid Aptamer-based Rapid Test Strips for the Detection of Staphylococcus aureus[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(11): 4577-4588.

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