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

Weiyei Zuo¹Congxue Shao¹He Qin¹Hongwei Gao²Xuemei Sun²Pengyan Wang^1*Jingjing Ren^1*

• ¹Shihezi University College of Animal Science and Technology, Shihezi, China
• ²Xinjiang Taikun Group Co., LTD, Changji, China

To address the need for rapid detection of Salmonella pullorum (S. pullorum) and Salmonella typhimurium (S. 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 ipaj gene of S. pullorum and the STM4497 gene of S. typhimurium, the optimized assay achieved detection at 37°C within 20 minutes. The dual RAA-LFD assay showed exceptional specificity with no cross-reactivity toward non-target pathogens. Detection sensitivities reached 5.91 × 10¹ CFU/mL (S. typhimurium) and 2.37 × 102 CFU/mL (S. pullorum) in pure cultures. In contrast, genomic DNA detection achieved identical limits of 5.70 × 101 fg/μL (S. typhimurium) and 4.53 × 101 fg/μL (S. pullorum). In artificially contaminated samples, the detection limits reached 3.92 × 102 CFU/mL for S. pullorum and 6.26 × 101 CFU/mL for S. 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.