A Pilot Pangenome-Based Screen of Ten Campylobacter jejuni Genomes Reveals Candidate Genus-Specific Diagnostic Markers

Campylobacter jejuni is a leading global cause of bacterial gastroenteritis, with significant economic and public health burdens. Conventional culture-based detection methods are very slow and often fail to identify non-jejuni or coli species. There is a pressing need for rapid, sensitive, and specific molecular diagnostics, which relies on the identification of conserved, genus-specific genetic markers. As a first step toward this goal, we present a pilot-scale pangenome analysis designed to generate candidate markers for downstream validation. We conducted a pilot-scale pangenome analysis of a curated, geographically and source-diverse panel of 10 complete C. jejuni genomes from clinical and environmental sources across Bangladesh, China, and Europe. Genomes were assessed for quality, and a pangenome was constructed using Roary to categorize core and accessory genes. Phylogenetic relationships were inferred, and conserved protein families were analyzed. Putative genus-specific marker genes were identified through in-depth BLASTN analysis against the non-redundant nucleotide database. Primer pairs for these targets were designed in silico and validated for specificity and efficiency. The C. jejuni pangenome comprised 3,518 genes, characterized as an "open" pangenome with a core of 1,289 genes and a large accessory genome of 2,229 genes, indicating high genomic plasticity. Phylogenetic analysis revealed distinct geographical clustering of isolates. We identified five highly conserved protein-coding genes specific to the Campylobacter genus, including a methionine ABC transporter substrate-binding protein and a hemerythrin domain protein. BLASTN analysis confirmed 100% identity and coverage exclusively with Campylobacter sequences for most targets. Successful in silico primer design yielded specific primer sets with optimal properties for all five markers. This pilot study leverages pangenomics to identify a suite of candidate, highly specific genetic markers for the Campylobacter genus, warranting validation on larger genome collections. The in-silico validation of corresponding primers provides a robust foundation for developing rapid PCR-based assays. These findings address a critical gap in food safety and clinical diagnostics, offering a path towards improved detection and surveillance of this significant pathogen. Further wet-lab validation is recommended to confirm assay performance.