Rapid Detection of <i>Salmonella</i> and <i>Listeria monocytogenes</i> in Fresh Beef Using Loop-Mediated Isothermal Amplification

This study developed and optimized loop-mediated isothermal amplification (LAMP) assays for rapid detection of <i>Salmonella</i> and <i>L. monocytogenes</i> in fresh beef. LAMP primers targeting the <i>invA</i> gene of <i>Salmonella</i> and <i>hly</i> gene of <i>L. monocytogenes</i> were used. Reaction conditions including temperature, dNTP concentration, Mg²⁺ concentration, primer ratio, and Bst DNA polymerase amount were optimized for each pathogen. Differences were observed between the two pathogens in optimal temperature, Mg²⁺ concentration, and Bst DNA polymerase requirements, highlighting the importance of pathogen-specific optimization. The optimized assays demonstrated high sensitivity with detection limits of 120 fg/μL for <i>Salmonella</i> and 130 fg/μL for <i>L. monocytogenes</i>, achieving detection within 40 and 60 min, respectively. Specificity tests confirmed both assays were highly specific for their target pathogens in fresh beef samples with no cross-reactivity observed. Addition of hydroxynaphthol blue enabled simple visual detection of positive results, eliminating the need for specialized equipment. The developed LAMP assays offer rapid, sensitive, and specific detection of these important foodborne pathogens.