Evaluating the Anti-Salmonella Aptamer Using Bioinformatic Tools and Enzyme-linked Aptamer Assay Method

Background: Identifying and quantifying food pathogens and environmental samples requires accurate, high-sensitivity, and specific methods. Salmonella is a zoonotic bacterium that can directly cause human diseases. This work aims to develop anti-Salmonella aptamer using bioinformatic tools and enzyme-linked aptamer assay (ELAA) to identify Salmonella and assess its affinity to simulated protein. Materials and Methods: An aptamer sequence was chosen to attach to Salmonella’s surface protein (outer membrane protein A [OmpA]). Then, the aptamer’s secondary and tertiary structures were achieved using the UNAFOLD and ROSETTA servers. The structure of OmpA was simulated by two methods: ab initio (using I-TASSER online server) and homology modeling (using MODELLER software and molecular dynamics simulation). In the next step, aptamer-protein binding was assessed via HDOCK software. Afterward, aptamer and biotin-labeled primers were synthesized to amplify specific sequences. Finally, the performance of aptamer to detect Salmonella was assessed by streptavidin conjugated with horseradish peroxidase (SA-HRP) using the ELAA method. Results: This study showed that the aptamer binds to OmpA and is acceptable with a -283.3 docking score. The superiority of the selected aptamer to random aptamers was also reviewed and approved. After assuring the validation of an aptamer with bioinformatic analysis, the aptamer was cloned in the pTZ57R plasmid and then used as a template in PCR. After the optimization of PCR and ELAA, aptamer performance was successfully performed to identify Salmonella. Conclusion: The designed aptasensor can be used for future investigations to detect Salmonella.