INIAV - National Institute for Agrarian and Veterinarian Research, Rua dos Lagidos, Vairão, 4485-655, Vila do Conde, Portugal.
LEPABE - Laboratory for Process Engineering, Environment, Biotechnology and Energy, Faculty of Engineering, University of Porto, Rua Dr. Roberto Frias, 4200-465, Porto, Portugal.
Sci Rep. 2024 May 18;14(1):11345. doi: 10.1038/s41598-024-61094-3.
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 (K) 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.
金黄色葡萄球菌肠毒素 A（SEA）是引起葡萄球菌食物中毒（SFP）爆发的最常见原因。适体是单链核酸，在包括诊断在内的多个领域被视为抗体的有前途替代品。在这项工作中，指数富集配体系统进化（SELEX）被用于筛选针对 SEA 的 DNA 适体。SELEX 方案使用磁珠作为目标分子的固定基质，并使用实时定量 PCR（qPCR）进行监测和优化序列富集。经过 10 轮选择，具有最高亲和力的 ssDNA 池通过下一代测序（NGS）进行测序。鉴定出大约 300 万个适体候选物，并选择最具代表性的簇序列进行进一步表征。亲和力最高的适体显示实验解离常数（K）为 13.36±18.62 nM。温度升高对适体与靶标的亲和力产生负面影响。在侧向流动测定中应用所选适体证明了它们在检测含有 100ng SEA 的样品中的功能，SEA 的最低含量足以引起食物中毒。总体而言，DNA 适体在 SEA 识别中的适用性在不同条件下得到了证明和表征，为开发诊断工具铺平了道路。
