Department of Microbiology, Defence Food Research Laboratory, Siddarthanagar, Mysuru, Karnataka, India; Defence Food Research Laboratory (DFRL), Mysuru, Karnataka State, 570011, India.
Department of Microbiology, Defence Food Research Laboratory, Siddarthanagar, Mysuru, Karnataka, India.
Anal Biochem. 2022 Nov 1;656:114884. doi: 10.1016/j.ab.2022.114884. Epub 2022 Sep 5.
S. Typhimurium is the most common food-borne pathogen frequently encountered in contaminated food and poses a major threat to public health, animals, and the food industry worldwide. Early and sensitive detection is the prime step to ensuring the microbiological quality of fresh and processed food. We used the cross-over SELEX strategy to select ssDNA aptamers against OM usher protein, FimD that could be employed in the S. Typhimurium detection assay. Altogether, 7 rounds of protein-SELEX, 3 rounds of intermittent counter-SELEX, and 4 rounds of whole bacterial cell-SELEX were undertaken for specific aptamer selection. The aptamer specificity was achieved by performing counter-SELEX with non-target bacterial cells. The enriched pool of aptamers obtained post SELEX process was cloned, sequenced, and further the specificity and affinity were characterized by flow cytometry. The aptamers DFRM-3 and DFRM-10 were specific and showed relatively high binding affinity towards S. Typhimurium with an apparent dissociation constant (Kd value) of 76 nM and 90.01 nM respectively. A dual aptamer-based sandwich assay was developed using biotinylated DFRM-3 aptamer for magnetic capture and AlexaFluor-488 labeled DFRM-10 aptamer for detection of S. Typhimurium. The limit of detection (LOD) of S. Typhimurium by the developed assay was found to be 10 CFU/ml in pure culture and 10 CFU/ml in the artificially contaminated chicken meat samples.
肠炎沙门氏菌是最常见的食源性病原体，常存在于污染的食物中，对全球公共卫生、动物和食品工业构成重大威胁。早期、灵敏的检测是确保新鲜和加工食品微生物质量的首要步骤。我们使用交叉 SELEX 策略筛选针对 OM usher 蛋白、FimD 的 ssDNA 适体，可用于沙门氏菌检测分析。总共进行了 7 轮蛋白 SELEX、3 轮间歇反 SELEX 和 4 轮全细菌细胞 SELEX，以进行特异性适体选择。通过用非靶标细菌细胞进行反 SELEX 实现适体特异性。SELEX 后获得的富集适体池被克隆、测序，并通过流式细胞术进一步表征其特异性和亲和力。适体 DFRM-3 和 DFRM-10 具有特异性，对肠炎沙门氏菌显示出相对较高的结合亲和力，表观解离常数（Kd 值）分别为 76 nM 和 90.01 nM。使用生物素化 DFRM-3 适体进行磁性捕获和 AlexaFluor-488 标记的 DFRM-10 适体用于检测肠炎沙门氏菌，开发了基于双重适体的夹心测定法。开发的测定法检测纯培养物中肠炎沙门氏菌的检测限（LOD）为 10 CFU/ml，人工污染鸡肉样品中的检测限为 10 CFU/ml。
