A novel multiplex PCR based method for the detection of Listeria monocytogenes clonal complex 8

Listeria monocytogenes is an important foodborne pathogen worldwide, which could cause listeriosis with a 20-30 % fatality rate in immunocompromised individuals. Listeria monocytogenes MLST clonal complex (CC) 8 strain is a common clone in food and clinical cases. The aim of this study was to develop multiplex PCR (mPCR) and high-resolution melting (HRM) qPCR to simultaneously detect L. monocytogenes CC8 and the other L. monocytogenes strains based on pan-genome analysis. A novel multiplex PCR and HRM qPCR targeted for the genes LM5578_1180 (specific for CC8) and LM5578_2262 (for L. monocytogenes) were developed. The specificity of this multiplex PCR and HRM qPCR were verified with other CCs of L. monocytogenes and other species strains. The detection limit of this multiplex PCR and HRM qPCR is 2.1 × 10³ CFU/mL and 2.1 × 10⁰ CFU/mL, respectively. This multiplex PCR and HRM qPCR could accurately detect CC8 strains with the interference of different ratios of L. monocytogenes CC9, CC87, CC121, CC155, and L. innocua strains. Subsequently, the detection ability of mPCR and HRM qPCR were also evaluated in spiked samples. The mPCR method could successfully detect 6.2 × 10³ CFU/mL of CC8 L. monocytogenes after 6 h enrichment while the multiplex HRM qPCR method could successfully detect 6.2 × 10⁴ CFU/mL of CC8 L. monocytogenes after 3 h enrichment. The feasibility of these methods were satisfactory in terms of sensitivity, specificity, and efficiency after evaluating 12 mushroom samples and was consistent with that of the National Standard Detection Method (GB4789.30-2016). In conclusion, the developed assays could be applied for rapid screening and detection of L. monocytogenes CC8 strains both in food and food production environments, providing accurate results to adopt monitoring measures to improve microbiological safety.