Campylobacter spp. are the causative agents of campylobacteriosis, a major foodborne illness globally, with millions of cases reported annually. These pathogens pose significant risks to both human and animal health. Conventional culture-based diagnostic methods are labor-intensive and time-consuming, underscoring the need for more efficient molecular detection strategies. This study employed a pangenomic analysis to identify novel gene-specific markers for pathogenic Campylobacter species and subspecies, laying the groundwork for their application in diverse diagnostic assays. A curated dataset of 105 high-quality genomes, representing 33 species and 9 subspecies, was analyzed using the Roary ILP Bacterial Annotation Pipeline. The results revealed substantial genomic diversity within the genus, with core gene counts varying across different nucleotide identity thresholds. Ribosomal genes such as rpsL, rpsJ, rpsS, rpmA, rpsK, rpsU, rpsG, rpmH, and rpsZ were consistently identified in the core genome, whereas accessory genes exhibited marked variability. This study uncovered novel and highly specific genetic markers for various Campylobacter species, including petB, clpX, and carB for C. coli; hypothetical proteins for C. jejuni and C. fetus; porA2 for C. lari; and mdtJ for C. upsaliensis. These markers demonstrated a specificity of at least 90% with minimal cross-reactivity with non-target organisms. The findings underscore the genomic heterogeneity within Campylobacter and provide essential genetic targets for the enhanced molecular detection of its pathogenic species, subspecies, and biovars.

Shiga toxin-producing Escherichia coli (STEC) are genetically diverse foodborne pathogens of major global public health concerns. Serogroup-level identification is critical for effective surveillance and outbreak control; however, it is often challenged by STEC’s genome plasticity and frequent recombination. In this study, we employed a standardized pangenomic pipeline integrating Roary ILP Bacterial Core Annotation Pipeline (RIBAP) and Panaroo to analyze 160 complete, high-quality STEC genomes representing eight major serogroups at a 95% sequence identity threshold. Candidate serogroup-specific markers were identified using gene presence/absence profiles from RIBAP and Panaroo. Our analysis revealed several high-confidence markers, including metabolic genes (dgcE, fcl_2, dmsA, hisC) and surface polysaccharide-related genes (capD, rfbX, wzzB). Comparative pangenomic evaluation showed that RIBAP predicted a larger pangenome size than Panaroo. Additionally, some genomes from the O104:H1, O145:H28, and O45:H2 serotypes clustered outside their expected clades, indicating sporadic serotype misplacements in phylogenetic reconstructions. Functional annotation suggested that most candidate markers are involved in critical processes such as glucose metabolism, lipopolysaccharide biosynthesis, and cell surface assembly. Notably, approximately 22.9% of the identified proteins were annotated as hypothetical. Overall, this study highlights the utility of pangenomic analysis for potential identification of clinically relevant STEC serogroups markers and phylogenetic interpretation. We also note that pangenome analysis could guide the development of more accurate diagnostic and surveillance tools.

Salmonella remains a leading cause of foodborne illness worldwide, with poultry products representing a major source of human exposure, underscoring the need for rapid and reliable detection methods. Although qPCR offers sensitive and timely pathogen detection, assay performance is highly dependent on sample preparation efficiency and nucleic acid purity, particularly in complex food matrices. In this study, we systematically optimized the sample preparation workflow of a SYBR Green based qPCR assay for enrichment-free detection of Salmonella enterica in poultry. Multiple lysis chemistries, incubation times, DNA extraction methods, centrifugation strategies, inoculum sources, and magnetic nanoparticle (MNP) assisted workflows were evaluated using phosphate-buffered saline and chicken rinsate matrices. Among the conditions tested, lysis with 20 µL Proteinase K and 400 µL PrepMan™ for 20 min produced the lowest and most consistent Cq values. Although Promega Wizard^® produced slightly lower mean Cq values than PrepMan™, statistical analysis showed no significant differences between extraction methods or centrifugation protocols, indicating comparable overall performance. Broth-derived inocula yielded earlier and more reproducible Cq values than colony-derived preparations. In contrast, inclusion of MNP processing resulted in higher Cq values in both matrices compared to the non-MNP workflow. Overall, these findings demonstrate that optimized lysis, extraction, and centrifugation workflows enhances the consistency and analytical reliability of direct qPCR detection of Salmonella in poultry matrices, supporting laboratory-based rapid detection applications.

Rapid detection and quantification of epidemiologically important Salmonella enterica serovars are critical for poultry surveillance, food safety monitoring, and risk-based intervention strategies. This study performed comprehensive analytical validation together with a preliminary field-based diagnostic performance evaluation of GenoPATHX™, a multiplex probe-based qPCR platform designed for the simultaneous detection and quantification of priority Salmonella serovars in poultry-associated matrices. The platform consists of two multiplex panels, designated the Chicken Key Performance Indicator (CKPI) and Turkey Key Performance Indicator (TKPI), each designed to detect priority poultry-associated Salmonella serovars together with a genus-level S. enterica marker. Analytical performance was evaluated for amplification efficiency, linearity, limit of detection (LoD₉₅), limit of quantification (LoQ), repeatability, intermediate precision, analytical specificity (inclusivity/exclusivity), robustness, matrix effects, and performance in artificially inoculated matrices. Diagnostic performance was further assessed using naturally contaminated poultry environmental samples. The assay demonstrated robust amplification performance in both singleplex and multiplex formats, with high linearity (R² = 0.987–0.999) and LoD₉₅ values ranging from 60 to 545 genome equivalents per reaction. Complete analytical inclusivity and high exclusivity were achieved for the evaluated isolate panel. In field samples, the direct GenoPATHX™ workflow demonstrated 81.0% sensitivity, 91.3% specificity, and substantial agreement with the USDA-FSIS reference culture method (κ = 0.73). Overall, GenoPATHX™ exhibited robust analytical performance and enabled rapid, same-day quantitative detection of priority Salmonella serovars in poultry-associated matrices, supporting its application for poultry surveillance and food safety monitoring.

The genus Listeria comprises diverse bacteria with significant public health relevance, particularly Listeria monocytogenes. 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 60–95%. 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 ≥80% 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.