Development of a dual-target RAA-LFD assay for point-of-care and visual detection of Salmonella pullorum and Salmonella typhimurium in fecal samples

To address the need for rapid detection of <i>Salmonella</i> pullorum (<i>S.</i> pullorum) and <i>Salmonella</i> typhimurium (<i>S.</i> typhimurium) in the poultry industry, we developed a dual-target point-of-care system integrating recombinase-aided amplification (RAA) with lateral flow dipsticks (LFD) for visual pathogen identification (RAA-LFD). Using primers and probes specifically targeting the <i>ipaj</i> gene of <i>S.</i> pullorum and the <i>STM4497</i> gene of <i>S.</i> typhimurium, the optimized assay achieved detection at 37 °C within 20 min. The dual RAA-LFD assay showed exceptional specificity with no cross-reactivity toward non-target pathogens. Detection sensitivities reached 5.91 × 10¹ CFU/mL (<i>S.</i> typhimurium) and 2.37 × 10² CFU/mL (<i>S.</i> pullorum) in pure cultures. In contrast, genomic DNA detection achieved identical limits of 5.70 × 10¹ fg/μL (<i>S.</i> typhimurium) and 4.53 × 10¹ fg/μL (<i>S.</i> pullorum). In artificially contaminated samples, the detection limits reached 3.92 × 10² CFU/mL for <i>S.</i> pullorum and 6.26 × 10¹ CFU/mL for <i>S.</i> typhimurium. Clinical validation demonstrated 96.88-100% coincidence with biochemical identification and multiplex PCR results. This study confirms the precision and high sensitivity of the dual RAA-LFD assay as a detection methodology. Furthermore, by eliminating reliance on complex traditional techniques, this technology provides an efficient grassroots-level field screening tool with significant potential for preventing avian salmonellosis and enhancing food safety monitoring.