Development of ssDNA Aptamers for the Sensitive Detection of Salmonella typhimurium and Salmonella enteritidis
Development of ssDNA Aptamers for the Sensitive Detection of Salmonella typhimurium and Salmonella enteritidis
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DOI：
10.1007/s12010-014-1103-z
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发表时间：
2014-09-01
影响因子：
3
通讯作者：
Yoon, Moon-Young
中科院分区：
文献类型：
--
作者：
Park, Hae-Chul;Baig, Irshad Ahmed;Yoon, Moon-Young
肠道沙门氏菌肠道亚种肠道沙门氏菌（Salmonella enterica ser.）肠沙门氏菌属（enterica ser. typhimurium）是最常见和严重的食源性病原体，可引起人和动物的沙门氏菌病。开发早期和超灵敏的检测系统是控制这种疾病的第一个关键步骤。为了实现这一点，我们使用了细胞系统进化配体指数富集（Cell-SELEX）技术，以确定单链DNA（ssDNA）适体可用作检测探针，可以特异性地结合到S。肠炎沙门氏菌和S.鼠伤寒。通过10轮Cell-SELEX筛选，在严格的筛选条件下共获得12个靶特异性ssDNA适体，阴性筛选进一步提高了这些适体的选择性。用革兰氏阴性菌E.大肠杆菌和铜绿假单胞菌，并发现对S.肠炎沙门氏菌和S.鼠伤寒。重要的是，三个候选适体表现出更高的结合亲和力和解离常数（Kd）被发现是在纳摩尔至亚微摩尔水平的范围内。此外，将单个适体缀合到多价定向适体聚合物上，与单独的单个适体相比，这导致结合亲和力增加100倍。总之，本研究报告了较高的亲和力和特异性的ssDNA适配体（30聚体），这可能是有用的捕获和检测探针在生物传感器为基础的检测系统沙门氏菌的鉴定。
Salmonella enterica subsp. enterica ser. enteritidis and Salmonella enterica subsp. enterica ser. typhimurium are the most common and severe food-borne pathogens responsible for causing salmonellosis in humans and animals. The development of an early and ultra-sensitive detection system is the first critical step in controlling this disease. To accomplish this, we used the cell systematic evolution of ligands by exponential enrichment (Cell-SELEX) technique to identify single-stranded DNA (ssDNA) aptamers to be used as detection probes that can specifically bind to S. enteritidis and S. typhimurium. A total of 12 target-specific ssDNA aptamers were obtained through ten rounds of Cell-SELEX under stringent selection conditions, and negative selection further enhanced the selectivity among these aptamers. Aptamer specificity was investigated using the gram-negative bacteria E. coli and P. aeruginosa and was found to be much higher towards S. enteritidis and S. typhimurium. Importantly, three candidate aptamers demonstrated higher binding affinities and the dissociation constants (Kd) were found to be in the range of nanomolar to submicromolar levels. Furthermore, individual aptamers were conjugated onto polyvalent directed aptamer polymer, which led to 100-fold increase in binding affinity compared to the individual aptamers alone. Taken together, this study reports the identification of higher affinity and specificity ssDNA aptamers (30mer), which may be useful as capture and detection probes in biosensor-based detection systems for salmonellosis.