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基于全细胞 SELEX 和 FACS 分析筛选鉴定鼠伤寒沙门氏菌特异性 DNA 适体。

Identification of Salmonella Typhimurium-specific DNA aptamers developed using whole-cell SELEX and FACS analysis.

机构信息

Department of Agricultural Engineering, National Academy of Agricultural Sciences, Suwon 441-707, South Korea.

出版信息

J Microbiol Methods. 2013 Nov;95(2):162-6. doi: 10.1016/j.mimet.2013.08.005. Epub 2013 Aug 24.

Abstract

Conventional methods for detection of infective organisms, such as Salmonella, are complicated and require multiple steps, and the need for rapid detection has increased. Biosensors show great potential for rapid detection of pathogens. In turn, aptamers have great potential for biosensor assay development, given their small size, ease of synthesis and labeling, lack of immunogenicity, a lower cost of production than antibodies, and high target specificity. In this study, ssDNA aptamers specific to Salmonella Typhimurium were obtained by a whole bacterium-based systematic evolution of ligands by exponential enrichment (SELEX) procedure and applied to probing S. Typhimurium. After 10 rounds of selection with S. Typhimurium as the target and Salmonella Enteritidis, Escherichia coli and Staphylococcus aureus as counter targets, the highly enriched oligonucleic acid pool was sorted using flow cytometry. In total, 12 aptamer candidates from different families were sequenced and grouped. Fluorescent analysis demonstrated that aptamer C4 had particularly high binding affinity and selectivity; this aptamer was then further characterized.

摘要

传统的感染性生物(如沙门氏菌)检测方法比较复杂,需要多个步骤,而且对快速检测的需求也在增加。生物传感器在病原体的快速检测方面显示出巨大的潜力。反过来,由于适体体积小、合成和标记容易、免疫原性低、生产成本低于抗体以及具有高靶特异性,因此非常适合用于生物传感器分析的开发。在这项研究中,通过基于全细菌的指数富集配体系统进化(SELEX)程序获得了针对鼠伤寒沙门氏菌的 ssDNA 适体,并将其应用于探测鼠伤寒沙门氏菌。经过 10 轮以鼠伤寒沙门氏菌为靶标、肠炎沙门氏菌、大肠杆菌和金黄色葡萄球菌为对照靶标的选择后,使用流式细胞术对高度富集的寡核苷酸池进行分类。总共从不同家族中测序并分组了 12 个适体候选物。荧光分析表明,适体 C4 具有特别高的结合亲和力和选择性;然后对该适体进行了进一步的表征。

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