Development of Immunoassays for Foodborne Pathogenic Bacteria Detection Using PolyHRP for Signal Enhancement

The rapid and accurate detection of foodborne pathogens is essential for ensuring food safety. <i>Escherichia coli</i> O157:H7 (<i>E. coli</i> O157:H7) and <i>Salmonella Typhimurium</i> (<i>S. Typhimurium</i>) are major foodborne pathogenic bacteria that pose significant public health risks, highlighting the need for effective detection methods. In this study, highly sensitive double-antibody sandwich-based enzyme-linked immunosorbent assays (ELISAs) were developed for the rapid detection of <i>E. coli</i> O157:H7 and <i>S. Typhimurium</i>, utilizing a streptavidin-polymerized horseradish peroxidase (SA-PolyHRP)-based signal enhancement system. Systematic optimization was performed on key parameters, including the capture antibody concentration, detection antibody, and blocking agent. Compared to the method using SA-HRP, substitution with SA-PolyHRP significantly improved detection sensitivity, achieving limits of detection (LODs) of 1.4 × 10⁴ CFU/mL for <i>E. coli</i> O157:H7 and 6.0 × 10³ CFU/mL for <i>S. Typhimurium</i>, with sensitivity enhancements of 7.86-fold and 1.83-fold, respectively. Specificity tests confirmed no cross-reactivity with non-target or closely related pathogenic strains. The matrix effect was effectively mitigated through 10-fold and 100-fold dilutions for <i>E. coli</i> O157:H7 and <i>S. Typhimurium</i>, respectively. Both pathogens were successfully detected in beef samples spiked with 5 CFU after 5 h of incubation. This study demonstrates the effectiveness of PolyHRP-based signal enhancement for the highly sensitive and specific detection of foodborne pathogens, offering a promising approach for rapid food safety monitoring and public health protection.