Development of a dual-target RAA-LFD assay for point-of-care and visual detection ofpullorum andtyphimurium in fecal samples.

Abstract

To address the need for rapid detection ofpullorum (pullorum) andtyphimurium (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 thegene ofpullorum and thegene oftyphimurium, 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 × 10 CFU/mL (typhimurium) and 2.37 × 10 CFU/mL (pullorum) in pure cultures. In contrast, genomic DNA detection achieved identical limits of 5.70 × 10fg/μL (typhimurium) and 4.53 × 10fg/μL (pullorum). In artificially contaminated samples, the detection limits reached 3.92 × 10 CFU/mL forpullorum and 6.26 × 10 CFU/mL fortyphimurium. 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.