Development and validation of a rapid Listeria monocytogenes loop-mediated isothermal amplification assay in soft cheese and raw pet food

Introduction Rapid screening of Listeria monocytogenes in human and animal foods is imperative for improving laboratory efficiency and safeguarding food supply. Methods We developed a loop-mediated isothermal amplification (LAMP) assay targeting the L. monocytogenes hlyA gene (listeriolysin O) and performed single-laboratory validation in representative human (soft cheese) and animal (raw pet food) food matrices. This study followed the U.S. Food and Drug Administration’s (FDA) Microbiological Method Validation Guidelines and used the FDA’s Bacteriological Analytical Manual (BAM) method as a reference. Results The assay produced accurate results (positive and negative) by analyzing the inclusivity and exclusivity panels comprising 50 L. monocytogenes , 20 non- L. monocytogenes Listeria , and 31 non- Listeria , with a detection limit of 100 cells per reaction, when using L. monocytogenes American Type Culture Collection (ATCC) 19115. Both soft cheese and raw pet food validations yielded satisfactory results, with the relative levels of detection (RLODs) falling within the acceptability limit (1.5) for a paired study. This suggests that LAMP analysis of 48 h enrichments was as sensitive as the BAM culture method in detecting low-level L. monocytogenes contamination in both matrices, with reliable screening results available at least 3 days sooner. Discussion This study demonstrates the potential of this rapid and reliable LAMP assay for routine screening of L. monocytogenes in soft cheese and raw pet food and, upon further validation, additional human and animal foods to ensure product safety and protect public health.