Integrating phage recognition and ultrasensitive CRISPR/Cas12a for breakthrough detection of viable-but-non-culturable Listeria monocytogenes

Listeria monocytogenes is a major foodborne pathogen capable of entering a viable but non-culturable (VBNC) state under environmental stress, leading to false negatives in conventional detection methods and posing substantial risks to food safety. Phages, which selectively infect viable bacteria, represent ideal biological recognition elements for enhancing viability-based detection. This study developed a phage amplification-assisted CRISPR/Cas12a assay (PAA-CRISPR/Cas12a) for the sensitive and specific detection of L. monocytogenes , including VBNC cells. Using the Listeria phage vB-LmoM-NJ05 as the recognition element, which covers major pathogenic serotypes (1/2a, 1/2b, 4b) with a 70% lysis rate of 42 tested L. monocytogenes strains, the method exploits phage-specific infection and lysis of L. monocytogenes , the method exploits phage-specific infection and lysis of L. monocytogenes . Progeny phages released during infection were thermally lysed to release nucleic acids, followed by CRISPR/Cas12a-mediated target recognition and signal amplification. Key parameters, including co-culture duration and Cas12a/crRNA concentrations, were optimized to enhance detection performance. The assay achieved detection limits of 4.16 × 10 1 copies/mL for pure DNA and 3.1 × 10 1 CFU/mL for L. monocytogenes NJ05 within 8 h. In addition, no cross-reactivity was observed with other common foodborne pathogens, confirming its high specificity. Meanwhile, the assay exhibited robust detection in spiked lettuce matrices. Given its strong viability discrimination and high sensitivity, the PAA-CRISPR/Cas12a assay provides an effective strategy for monitoring L . monocytogenes contamination in foods and holds significant potential for rapid food safety testing. • Utilizing the Listeria phage vB-LmoM-NJ05 as the recognition element, we developed a phage amplification-assisted CRISPR/Cas12a assay (PAA-CRISPR/Cas12a) for the sensitive and specific detection of L. monocytogenes. • The PAA- CRISPR/Cas12a assay enables the specific detection of viable cells, including L. monocytogenes in a viable but non-culturable (VBNC) state. • This assay achieved detection limits of 4.16 × 10 1 copies/mL for pure DNA and 3.1 × 10 1 CFU/mL for L. monocytogenes within 8-hour timeframe. • The PAA- CRISPR/Cas12a assay exhibited robust detection in lettuce matrices, offering a rapid and cost-effective solution for food safety monitoring.