基于适配体拉曼光谱技术快速检测肠炎沙门氏菌方法

目的:应用SELEX技术筛选高亲和力、高特异性适配体,利用该适配体结合拉曼光谱技术建立肠炎沙门氏菌快速检测方法。方法:采用全细菌指数富集的配体系统进化技术(whole-bacteria systematic evolution of ligands by exponential enrichment,whole-bacteria-SELEX)筛选肠炎沙门氏菌特异性核酸适配体,并采用酶联免疫吸附实验(enzyme-linked immuno sorbent assay,ELISA)与SERS技术对筛选出的适配体亲和力及特异性进行评价,建立肠炎沙门氏菌检测方法。结果:本研究通过对肠炎沙门氏菌进行十五轮SELEX筛选,并通过ELISA对其亲和力进行评价,筛选出Aptamer₄、Aptamer₁₀、Aptamer₁₂三条候选适配体,并通过SERS技术确认Aptamer₄为亲和力最佳适配体;将Aptamer₄与肠炎沙门氏菌、肺炎克雷伯氏菌等5种混合菌结合,结果表明,通过SERS技术可特异的检测出肠炎沙门氏菌,且该方法重复性较好,其最低检测限的细菌浓度为102 CFU/mL。且在猪肉样品的检测中,肠炎沙门氏菌的回收率为93.37%~100.18%。结论:应用SELEX方法成功筛选出与肠炎沙门氏菌高特异性、高亲和力适配体,并建立基于表面增强拉曼光谱技术快速检测肠炎沙门氏菌的方法,该方法特异性强、灵敏度高、成本低、快速简便,可应用于食品加工过程中肠炎沙门氏菌的快速检测。

Rapid Detection of Salmonella enteritidis Based on Aptamer Raman Spectroscopy

Objectives:SELEX technology was used to screen high affinity and high specificity aptamer,and the rapid detection method of Salmonella enteritidis was established by using this aptamer combined with Raman spectroscopy. Methods:The specific aptamers of Salmonella enteritidis were screened by the ligand system evolution technology with bacterial index enrichment. The affinity and specificity of the aptamers were evaluated by enzyme-linked immunosorbent assay and SERS technology,and the detection method of Salmonella enteritidis was established. Results:In this study,aptamer₄,aptamer₁₀,and aptamer₁₂ as three candidate aptamers were selected through 15 rounds of SELEX screening for Salmonella enteritidis,and their affinity were evaluated by ELISA. Aptamer₄ was confirmed as the best aptamer by SERS technology. Aptamer₄ was combined with 5 kinds of mixed bacteria such as Salmonella enteritis and Klebsiella pneumoniae. The results showed that Salmonella enteritidis could be detected specifically and the repeatability of this method was good. The lowest detection limit of bacterial concentration was 102 CFU/mL. The recovery of Salmonella enteritidis was 93.37%~100.18%. Conclusion:SELEX method was used to screen out the suitable ligands with high specificity and affinity for Salmonella enteritidis,and a rapid detection method for Salmonella enteritidis based on surface enhanced Raman spectroscopy was established. This method had strong specificity,high sensitivity,low cost,fast and simple,so that it could be used for the rapid detection of Salmonella enteritidis in food processing.