Ultrasensitive point-of-care testing of salmonella in lettuce and milk samples using a recombinase polymerase amplification-based colorimetric/fluorescent dual-readout lateral flow assay

A portable dual-mode RPA-based LFA sensor was developed for Salmonella detection in lettuce and milk samples. The fimY gene of Salmonella was chosen as specific conserved sequences, their corresponding primers based on RPA conditions were designed and optimized, and respectively labeled with Biotin and FAM groups at 5′ end, then applied for efficient fimY gene fragments enrichment via RPA. Sandwich construction of streptavidin-RPA products-AuNPs@FAM antibody was generated on T-line, showing positive results within 10 min, colorimetric-fluorescent signals were captured by a smartphone and converted into gray intensities and fluorescence intensities, further enabling the quantification of Salmonella with detection limits of 10 cfu/mL and 1 cfu/mL for colorimetric and fluorescent modes, respectively, over a linear range of 10¹–10⁶ cfu/mL. The proposed strategy provided a valuable reference for Salmonella one-site detection, which would be utilized as a universal method for other food-borne pathogens detection by matching specific conserved sequences and primer designs.