Microbial factors, including bacteria, viruses, and other pathogens, are significant contributors to foodborne illnesses, posing serious food safety risks due to their potential for rapid growth and contamination. Listeria monocytogenes is one of the most common types of foodborne bacteria that can cause serious foodborne diseases or even fatalities. In this study, a novel nucleic acid amplification method called Proofman-LMTIA was employed to detect Listeria monocytogenes contamination in food. This method combines proofreading enzyme-mediated probe cleavage with ladder-shape melting temperature isothermal amplification. A positive recombinant plasmid was used as a control to ensure the accuracy of the detection results, and primers and Proofman probes were specifically designed for the LMTIA. Genomic DNA was extracted, the reaction temperature was optimized, and the primers' specificity was verified using foodborne pathogens like Staphylococcus aureus, Escherichia coli O157:H7, and Salmonella. The sensitivity was assessed by testing serial dilutions of genomic DNA, and the method's applicability was confirmed by detecting artificially contaminated fresh pork. The established LMTIA method exhibited both high specificity and sensitivity. At the optimal reaction temperature of 63 °C, the primers specifically identified Listeria monocytogenes contamination in pork at a concentration of 8.0 ± 0.7 colony-forming units (CFUs) per 25 g. Furthermore, the Proofman-LMTIA method was applied to test Listeria monocytogenes DNA in 30 food samples purchased from a Chinese retail market, and reassuringly, all results indicated no contamination. Proofman-LMTIA can serve as a reliable and rapid method for detecting Listeria monocytogenes in food, contributing to public health by safeguarding consumers from foodborne illnesses, and strengthening food safety regulations.
Keywords: Listeria monocytogenes; Proofman probes; foodborne pathogens; ladder-shape melting temperature isothermal amplification.