PDF(Pubmed)
Abstract：
Staphylococcal enterotoxin A (SEA) is the most frequently reported in staphylococcal food poisoning (SFP) outbreaks. Aptamers are single-stranded nucleic acids that are seen as promising alternatives to antibodies in several areas, including diagnostics. In this work, systematic evolution of ligands by exponential enrichment (SELEX) was used to select DNA aptamers against SEA. The SELEX protocol employed magnetic beads as an immobilization matrix for the target molecule and real-time quantitative PCR (qPCR) for monitoring and optimizing sequence enrichment. After 10 selection cycles, the ssDNA pool with the highest affinity was sequenced by next generation sequencing (NGS). Approximately 3 million aptamer candidates were identified, and the most representative cluster sequences were selected for further characterization. The aptamer with the highest affinity showed an experimental dissociation constant (KD) of 13.36 ± 18.62 nM. Increased temperature negatively affected the affinity of the aptamer for the target. Application of the selected aptamers in a lateral flow assay demonstrated their functionality in detecting samples containing 100 ng SEA, the minimum amount capable of causing food poisoning. Overall, the applicability of DNA aptamers in SEA recognition was demonstrated and characterized under different conditions, paving the way for the development of diagnostic tools.
摘要：
葡萄球菌肠毒素A（SEA）是葡萄球菌食物中毒（SFP）暴发中最常见的报道。适体是单链核酸，在几个领域被视为抗体的有希望的替代品。包括诊断。在这项工作中,通过指数富集（SELEX）配体的系统进化用于选择针对SEA的DNA适体。SELEX方案采用磁珠作为靶分子的固定基质和实时定量PCR(qPCR)用于监测和优化序列富集。经过10个选择周期,通过下一代测序（NGS）对具有最高亲和力的ssDNA池进行测序。确定了大约300万个适体候选物，选择最具代表性的簇序列进行进一步表征。具有最高亲和力的适体显示13.36±18.62nM的实验解离常数(KD)。升高的温度负面影响适体对靶标的亲和力。选择的适体在侧流测定中的应用证明了它们在检测含有100ngSEA的样品中的功能。导致食物中毒的最低限度。总的来说,在不同条件下证明和表征了DNA适体在SEA识别中的适用性，为诊断工具的开发铺平了道路。
