Development of an aptamer-based rapid diagnostic assay for Salmonella detection using whole-cell SELEX technique

ABSTRACT Rapid and accurate detection of Salmonella is essential for improving food safety and reducing the burden of enteric infections, especially in low-resource settings. Aptamer-based diagnostic platforms offer a promising alternative to antibody-based methods, given their high specificity, stability, and low production cost. A DNA aptamer targeting Salmonella enterica was developed through Systematic Evolution of Ligands by Exponential Enrichment (SELEX) iterative rounds of selection. Target-binding efficiency was monitored by colorimetric and spectrophotometric binding tests. Silver nanoparticles (AgNPs) were synthesized and conjugated with candidate aptamers through direct adsorption. Dot blot colorimetric assays were performed by exposing aptamer–AgNP complexes to serial dilutions of Salmonella cultures ranging from 10² to 10⁸ CFU/mL. Aggregation was evaluated visually on nitrocellulose membranes and quantified by a UV-Vis spectrophotometer based on absorbance intensity and peak wavelength shifts. Controls included AgNPs alone and related non-target bacterial ( Escherichia coli ). Final aptamer pools were sequenced using Nextseq 550 Illumina platform to determine specific sequences and predict candidate secondary structures for future mass production. Aptamer selection showed progressive enrichment, with significant dot blot signal and aggregation from round 8, and strong binding after 12 rounds of SELEX. Upon exposure to Salmonella , aptamer–AgNP complexes exhibited a concentration-dependent aggregation response, with visual detection observed at bacterial concentrations of 10 5 CFU/mL and selective binding toward Salmonella compared to E. coli and spectrophotometrically quantifiable from 10⁵ CFU/mL. Peak absorbance increased from 0.31 AU for 10² CFU/mL target concentration to 1.73 AU with 10⁸ CFU/mL, and the wavelength shifted from 504 nm to 525 nm, indicating nanoparticle aggregation. No specific aggregation or spectral shifts were observed in control samples. The enriched aptamer candidates were identified and prioritized, exhibiting conserved secondary structures, including hairpins, multi-branched loops, and internal bulges, predicting to facilitate specific interactions with Salmonella outer membrane proteins or LPS-O antigen. This study demonstrates the diagnostic applicability of aptamer conjugated with AgNP colorimetric assays for sensitive and specific detection of Salmonella . Future work should focus on sequence-based chemical modifications to enhance aptamer immobilization and assay performance, thereby facilitating adaptation of the platform to lateral flow formats during mass production for point-of-care applications. IMPORTANCE This study addresses a critical need for rapid, low-cost, and sensitive diagnostic tools for Salmonella detection, particularly in low-resource settings where conventional methods are often inaccessible or time-consuming. By developing an aptamer-based colorimetric assay using silver nanoparticles, the research offers a highly specific, sensitive, and affordable alternative to existing diagnostics. The use of DNA aptamers selected through Systematic Evolution of Ligands by Exponential Enrichment enables precise recognition of Salmonella surface markers, while the colorimetric readout allows for easy visual detection without the need for sophisticated instruments. The assay’s sensitivity demonstrates its practical utility for early and field-level pathogen detection. Importantly, the incorporation of next-generation sequencing in aptamer characterization enhances the molecular understanding of aptamer–target interactions and supports future assay refinement for application in lateral flow assay. The findings support translating the platform into point-of-care formats, which help food safety monitoring and infectious disease control in developing regions.