Development of an IMSA assay targeting the hly gene for detection of Listeria monocytogenes in food samples.

Listeria monocytogenes remains a foodborne pathogen of major global public health concern, associated with high mortality, particularly in developing countries where food safety surveillance infrastructure is limited. The hly gene, which encodes the pore-forming toxin listeriolysin O, is a highly conserved virulence determinant across all major serotypes. In this study, we developed a closed-tube isothermal multiple self-matching-initiated amplification (IMSA) assay targeting the hly gene for the specific and visual detection of L. monocytogenes. Optimized to run at 63 °C for 45 min, the assay employs SYBR Green I preloaded in the tube cap, enabling direct visual readout under UV light without the post-amplification tube opening-a feature critical for resource-limited laboratories. The assay detected all six L. monocytogenes strains tested (serotypes 1/2a, 1/2c, 4b, and 4e) without cross-reactivity against 11 non-target foodborne pathogens. In phosphate-buffered saline (PBS), the limit of detection (LOD) was 1 × 102 CFU/mL, matching quantitative real-time PCR (qPCR) and outperforming loop-mediated isothermal amplification (LAMP) tenfold. This analytical sensitivity was determined in PBS; complex food matrices may elevate the practical detection limit owing to the presence of inhibitory substances. When applied to 84 enriched retail food samples from Anhui Province, China-spanning dairy, meat, and seafood-the assay identified 17 positive samples (20.24%) and showed complete concordance with the culture-based reference method. This closed-tube IMSA assay provides a rapid, specific, and equipment-minimal screening tool suitable for food safety monitoring in basic laboratories, particularly in developing regions lacking access to qPCR instrumentation.