A novel two-stage enzyme substrate assay for rapid screening and detection of Listeria monocytogenes in milk and dairy products

The contamination of milk and milk products with Listeria monocytogenes has been widely reported globally. This pathogen with a high case-fatality rate of up to 30 %, poses a serious public health concern. Conventional methods for its detection are time-consuming and laborious. Thus, the current research project was undertaken to develop novel two-stage enzyme-based assay for the rapid and selective detection of Listeria monocytogenes in milk and dairy products. In Stage I, the Listeria Selective Enrichment Medium (LSEM) supported the growth of Listeria spp. while suppressing a broad range of background microflora, with presumptive detection achieved via β-D-glucosidase-mediated black color development within 24 h at initial loads as low as 1.35 log cfu/mL. Stage II employed an Enzyme Substrate Medium (ESM) for metabolic confirmation, wherein L. monocytogenes, L. innocua, and L. ivanovii consistently yielded a characteristic green color within 3–4 h. The assay demonstrated strong selectivity, effectively inhibiting E. faecalis, S. aureus, B. cereus, lactic acid bacteria, and Gram-negative contaminants at low to moderate loads, while avoiding false positives even in the presence of high background contamination. Sensitivity studies confirmed a detection limit of ∼1.35 log cfu/mL, with successful recovery from spiked milk within 28 h. Evaluation of 95 milk and dairy product samples revealed L. monocytogenes in two pasteurized milk samples (2.1 % prevalence), with complete agreement between the developed assay and the ISO 11290–1:2017 method. Compared to conventional enrichment protocols, the two-stage approach offered superior inhibitory control over competing flora, minimized assay interference, and reduced detection time by enabling confirmation within ∼28 h. This method provides a rapid, specific, and cost-effective tool for L. monocytogenes surveillance in complex dairy matrices, with potential application in routine food safety monitoring and regulatory screening.