K-mer based method for finding sequence type-specific PCR primers for Listeria monocytogenes

Listeria monocytogenes is a major foodborne pathogen for which rapid strain typing is critical for outbreak investigation and food safety surveillance. Here we present a k-mer based workflow for identifying genomic regions unique to specific sequence types and converting them into practical PCR assays. Dataset of 28,545 public isolates were screened to discover candidate markers for 17 sequence types and two clonal complexes, and the resulting primer sets were evaluated on 51 isolates representing 17 sequence types in both singleplex and multiplex PCR formats. The workflow identified sequence type-specific k-mers with a median of 47,000 per target and yielded 83 primer pairs for experimental testing. In singleplex assays, at least one primer pair achieved complete discrimination for 15 of 17 targets. Three 5-plex panels retained robust target resolution, and the 15-plex assay generated the expected target-associated products across all 51 isolates, demonstrating that the selected primer pairs remained functional when combined. Overall, these results show that population genomic data can be translated into rapid, scalable, and low-cost PCR assays for targeted L. monocytogenes typing, providing a practical bridge between genomic surveillance and routine laboratory screening.