A rapid portable assay for Salmonella typhimurium quantitative detection based on performance-enhancing anti-matrix interference aptamer

Background

Salmonella typhimurium (S. typhimurium) is a common cause of global foodborne illness, and poses a significant risk to food safety and public health. Consequently, there is a pressing need to develop a portable, rapid, quantitative and highly sensitive detection method for S. typhimurium to enable real-time field detection. Although aptamer-based detection methods for S. typhimurium have garnered increasing attention, challenges remain in improving their sensitivity, portability, and quantification accuracy. Additionally, the use of portable instruments for rapid quantitative detection is essential for successfully applying rapid testing methods in field-based quantitative analysis.

Results

In this study, two truncation strategies were employed to optimize the binding properties of the aptamer ROU-77 while maintaining its resistance to matrix interference. This optimization yielded a truncated aptamer, ROU-46, which exhibited a 4.2-fold increase in target affinity compared to ROU-77. Based on these findings, a novel variant probe which incorporated the aptamer and DNA bridges was designed to develop a signal amplified portable high affinity aptamer assay. The target concentration was converted into measurable glucose levels via the formation of DNA bridges, with signal amplification achieved by modulating the number of these bridges. Consequently, a portable glucose meter enabled highly sensitive, quantitative detection of S. typhimurium. Under optimized conditions, the assay achieved a detection limit of 295 CFU⋅mL⁻¹. In addition, the excellent specificity, reproducibility, and practical applicability of this proposed method were carefully evaluated.

Significance

This study successfully truncated an anti-matrix interference aptamer and enhanced its affinity without compromising matrix resistance, which provided a novel strategy for aptamer optimization. Additionally, a new on-site detection method for S. typhimurium was developed by integrating the truncated aptamer into a composite probe and coupling it with a portable glucose meter. The use of the portable quantification device eliminated the need for large-scale equipment, and provided an innovative approach for real-time rapid detection.