Fallah Arezoo, Fooladi Abbas Ali Imani, Sedighian Hamid, Taheri Ramezan Ali, Behzadi Elham, Havaei Seyed Asghar
Department of Bacteriology and Virology, Faculty of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran.
Applied Microbiology Research Center, Biomedicine Technologies Institute, Baqiyatallah University of Medical Sciences, Tehran, Iran.
Int J Biol Macromol. 2025 May;307(Pt 1):140567. doi: 10.1016/j.ijbiomac.2025.140567. Epub 2025 Jan 31.
Staphylococcus aureus (S. aureus) is a globally prevalent foodborne pathogen responsible for significant public health concerns. Staphylococcal food poisoning (SFP) results from staphylococcal enterotoxins (SEs) produced by specific strains of S. aureus. Rapid and effective detection of SEs remains a significant challenge for public health authorities. Aptamers, short single-stranded DNA(ssDNA), RNA, or synthetic xeno nucleic acid (XNA) molecules, exhibit high affinity for binding to their specific targets. Due to their unique properties, including low production costs, ease of chemical modification, high thermal stability, and reproducibility, aptamers present a viable alternative to antibodies for diagnostic and therapeutic applications.
This research aimed to isolate high-affinity ssDNA aptamers with specificity for staphylococcal enterotoxin D (SED).
The systematic evolution of ligands by the exponential enrichment (SELEX) method was utilized to identify specific aptamers. These aptamers were then validated using enzyme-linked apta-sorbent assay (ELASA) and surface plasmon resonance (SPR) to assess their binding characteristics and affinity.
SELEX successfully identified aptamers with strong binding affinity to SED. Among the identified candidates, one aptamer, Aptamer 1, exhibited the highest specificity for SED with a dissociation constant (K) of 4.4 ± 2.26 nM. The limit of detection (LOD) for SED using this aptamer was determined to be 45 nM.
The findings indicate that the ELASA system designed using the aptamer developed in this study demonstrates higher specificity, sensitivity, and reproducibility in detecting enterotoxin D. This novel aptamer offers significant potential for applications in diagnostic platforms targeting S. aureus enterotoxins.
金黄色葡萄球菌是一种全球普遍存在的食源性病原体，引起了重大的公共卫生问题。葡萄球菌食物中毒（SFP）是由特定金黄色葡萄球菌菌株产生的葡萄球菌肠毒素（SEs）引起的。快速有效地检测SEs仍然是公共卫生当局面临的重大挑战。适体是短的单链DNA（ssDNA）、RNA或合成的异源核酸（XNA）分子，对其特定靶标具有高亲和力。由于其独特的性质，包括低成本生产、易于化学修饰、高热稳定性和可重复性，适体为诊断和治疗应用提供了一种可行的抗体替代物。
本研究旨在分离对葡萄球菌肠毒素D（SED）具有特异性的高亲和力ssDNA适体。
利用指数富集配体系统进化（SELEX）方法鉴定特异性适体。然后使用酶联适体吸附测定（ELASA）和表面等离子体共振（SPR）对这些适体进行验证，以评估其结合特性和亲和力。
SELEX成功鉴定出与SED具有强结合亲和力的适体。在鉴定出的候选适体中，一种适体Aptamer 1对SED表现出最高的特异性，解离常数（K）为4.4±2.26 nM。使用该适体检测SED的检测限（LOD）确定为45 nM。
研究结果表明，使用本研究中开发的适体制备的ELASA系统在检测肠毒素D方面具有更高的特异性、灵敏度和可重复性。这种新型适体在针对金黄色葡萄球菌肠毒素的诊断平台应用中具有巨大潜力。
