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A comparative genomic analysis of ten representative Listeria species was conducted using 33 high-quality genome assemblies to investigate core and accessory genome dynamics and identify candidate diagnostic loci. Pangenome reconstruction was performed using the Roary Integer Linear Programming Bacterial Annotation Pipeline (RIBAP) to classify core, soft-core, and accessory genes, while average nucleotide identity (ANI) analysis assessed genomic relatedness across thresholds of 60\u201395%. Functional annotation of core and species-specific genes was conducted using Genome Annotation and Information Analysis (GAIA). Core genes were highly conserved and associated with essential cellular functions, whereas the accessory genome contributed to species-level diversification and ecological adaptation. Candidate molecular markers were derived from accessory genes and evaluated based on presence\/absence across genomes, retaining loci present in \u226580% of target strains and absent in non-target strains. Experimental validation of selected primers was performed using two L. monocytogenes reference strains (ATCC 19117 and ATCC BAA-679) with conventional PCR and gel electrophoresis to confirm expected amplicon sizes and specificity. These findings establish a genome-informed, specificity-driven framework for marker development and highlight the accessory genome as a valuable source of diagnostic loci, supporting accurate detection, epidemiological surveillance, and food safety monitoring.<\/jats:p>","DOI":"10.3390\/pathogens15040397","type":"journal-article","created":{"date-parts":[[2026,4,7]],"date-time":"2026-04-07T12:03:47Z","timestamp":1775563427000},"page":"397","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Genome-Informed Identification of Species-Specific Diagnostic Markers for Listeria Using Pangenome Analysis"],"prefix":"10.3390","volume":"15","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-0710-6214","authenticated-orcid":false,"given":"Viona","family":"Osei","sequence":"first","affiliation":[{"name":"Center for Food Animal Health, Food Safety and Defense, Department of Pathobiology, College of Veterinary Medicine, Tuskegee University, Tuskegee, AL 36088, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Emmanuel","family":"Kuufire","sequence":"additional","affiliation":[{"name":"Center for Food Animal Health, Food Safety and Defense, Department of Pathobiology, College of Veterinary Medicine, Tuskegee University, Tuskegee, AL 36088, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rejoice","family":"Nyarku","sequence":"additional","affiliation":[{"name":"Center for Food Animal Health, Food Safety and Defense, Department of Pathobiology, College of Veterinary Medicine, Tuskegee University, Tuskegee, AL 36088, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kingsley E.","family":"Bentum","sequence":"additional","affiliation":[{"name":"Center for Food Animal Health, Food Safety and Defense, Department of Pathobiology, College of Veterinary Medicine, Tuskegee University, Tuskegee, AL 36088, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tyric","family":"James","sequence":"additional","affiliation":[{"name":"Center for Food Animal Health, Food Safety and Defense, Department of Pathobiology, College of Veterinary Medicine, Tuskegee University, Tuskegee, AL 36088, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Asmaa","family":"Elrefaey","sequence":"additional","affiliation":[{"name":"Center for Food Animal Health, Food Safety and Defense, Department of Pathobiology, College of Veterinary Medicine, Tuskegee University, Tuskegee, AL 36088, USA"},{"name":"Department of Food Hygiene and Control, Faculty of Veterinary Medicine, University of Sadat City, Sadat City 32897, Egypt"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9722-5318","authenticated-orcid":false,"given":"Temesgen","family":"Samuel","sequence":"additional","affiliation":[{"name":"Center for Food Animal Health, Food Safety and Defense, Department of Pathobiology, College of Veterinary Medicine, Tuskegee University, Tuskegee, AL 36088, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Woubit","family":"Abebe","sequence":"additional","affiliation":[{"name":"Center for Food Animal Health, Food Safety and Defense, Department of Pathobiology, College of Veterinary Medicine, Tuskegee University, Tuskegee, AL 36088, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,4,7]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"85","DOI":"10.1038\/s43705-023-00293-x","article-title":"Comparative genomics unveils extensive genomic variation between populations of Listeria species in natural and food-associated environments","volume":"3","author":"Liao","year":"2023","journal-title":"ISME Commun."},{"key":"ref_2","doi-asserted-by":"crossref","unstructured":"Sibanda, T., and Buys, E.M. 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