Multiplex Real-Time PCR Detection of Salmonella enterica Serovars in Egg Matrices: A Comparative Genomics Approach...

Rapid and accurate detection of Salmonella enterica serovars is critical for food safety monitoring, given their frequent involvement in egg-related foodborne disease outbreaks. In this study, unique genetic biomarkers for S. enterica, S. enterica serovars Enteritidis, Typhimurium, and Thompson were identified through comparative genomic analyses. Corresponding primer-probe sets were designed and evaluated using multiplex real-time PCR. The assay achieved amplification efficiencies between 90.7% and 94.2%, with strong linear correlations (R² > 0.998), and demonstrated high sensitivity with a detection limit of approximately 20 copies/µL. Specificity tests confirmed the absence of cross-reactivity with nontarget strains. Stability was further validated under accelerated aging conditions, with serovar-specific targets maintaining consistent detection, although the S. enterica marker exhibited modest variability (CV > 5%) for 12 days. Interference assessments revealed minimal inhibitory effects from common laboratory media, whereas egg matrices caused pronounced inhibition, particularly egg yolk. Subsequent analyses identified yolk granules as the primary source of inhibition, potentially associated with phosvitin and proteases. Further, DNA extraction methods influenced detection outcomes. The column-based extraction improved performance at higher yolk concentrations, whereas rapid heating-based extraction showed advantages at lower concentrations by denaturing inhibitory proteins and lipids. Lastly, application to artificially inoculated egg samples confirmed robust detection at ≥3 log CFU/mL, despite higher Ct values compared to tryptic soy broth controls. Collectively, this multiplex assay enables rapid, sensitive, and specific detection of major Salmonella serovars, while highlighting the importance of tailoring DNA extraction strategies to overcome matrix-associated inhibition in eggs.