The detection of foodborne pathogens is a critical aspect of ensuring food safety. Traditional methods rely on time-intensive enrichment steps and pathogen-specific assays, extending testing timelines and limiting throughput. This study evaluates an enrichment-free, multiplexed pathogen detection workflow combining the Pathotrak system for bacterial separation and the Neogen Molecular Detection System (MDS) for detection. The workflow enables simultaneous detection of Salmonella, Escherichia coli O157, Listeria monocytogenes, Listeria spp., and Campylobacter in poultry samples, significantly reducing the time taken to screen samples requiring further evaluation. The system’s performance was assessed using inoculated chicken samples over a range of bacterial concentrations (10²–10⁸ CFU/mL). The MDS system demonstrated robust detection for most pathogens, with strong correlations between theoretical inoculation levels and MDS-calculated concentrations (R² > 0.85 for all pathogens). However, detection variability was observed at lower concentrations for Salmonella and Listeria monocytogenes. The system maintained high sensitivity and specificity, achieving a Cohen’s Kappa coefficient for E. coli and Campylobacter. This study highlights the potential of enrichment-free, multiplex detection to streamline food safety testing by reducing the time to results, enhancing efficiency, and providing reliable pathogen quantification across multiple targets.

Salmonella in raw poultry drives foodborne outbreaks and recalls, and process-control decisions increasingly turn on quantitative thresholds rather than presence/absence. Acting on a threshold in real time requires accurate quantification near the cutoff and a result within one work shift. Conventional enrichment-based enumeration meets neither: it adds hours of growth before detection, and the growth step biases the count toward faster-growing, unstressed cells. Near-threshold false positives also matter: products below the limit tests positive, triggering costly and avoidable holds and recalls. We evaluated a same-shift, enrichment-free workflow that couples the Pathotrak concentrator with real-time PCR (Bio-Rad iQ-Check Salmonella II) for quantifying Salmonella in raw poultry, with sample preparation in about 30 min instead of 6–24 h of enrichment. The evaluation spanned 313 independent inoculations across three laboratories, three calendar years (2023–2025), and multiple seasons, technicians, instruments, and chicken sources. A latent-truth framework propagated inoculation uncertainty into sensitivity (Se) and specificity (Sp) estimates and made the half-log gray zone around each cutoff tractable. At 10 CFU/g, the workflow achieved Se 100% (20/20; 95% CI 83.2–100.0%) for samples at least 0.5 log₁₀ above the threshold and Sp 94.4% (185/196; 95% CI 90.2–97.2%) below it; within the half-log gray zone, classification was Se 88.6% (95% CI 68.8–99.1%)/Sp 76.5% (95% CI 62.1–88.1%) (n = 77). In a cross-study comparison, gray-zone performance at 10 CFU/g fell within the range reported for MPN, with inoculation strain, chicken source, and plate-count method differing by site. At 1 CFU/g, the workflow returned a calibrated quantitative result within a single shift, which, to our knowledge, no enrichment-free real-time-PCR workflow has previously achieved without an incubation step. Samples more than 0.5 log₁₀ CFU/g above or below a threshold were classified correctly in 205/216 (94.9%) cases at 10 CFU/g and 109/117 (93.2%; 95% CI 87.0–97.0%) at 1 CFU/g, with classification uncertainty confined to the gray zone (Se 63.2% (95% CI 49.9–76.0%)/Sp 66.5% (95% CI 54.2–77.9%), n = 166) as expected at low copy number (Salmonella genome copies estimated by qPCR).