High-resolution melting curve real-time PCR for rapid differentiation of Escherichia coli and Campylobacter jejuni in poultry samples

Foodborne pathogens remain a major challenge for public health and food safety worldwide. Rapid and accurate molecular methods are essential for the early detection of bacterial contamination in food products. The present study evaluated the application of real-time PCR combined with melting curve analysis for the rapid differentiation of two major foodborne pathogens, Escherichia coli and Campylobacter jejuni. A total of 86 chicken carcass swab samples were collected from poultry markets in Baghdad, Iraq. Specific primers targeting the yhaV gene of E. coli and the hipO gene of C. jejuni were used in a real-time PCR assay followed by melting curve analysis. Distinct melting temperature profiles were obtained for each species. The mean melting temperature for E. coli isolates was 79.75 ± 0.29 °C, whereas C. jejuni isolates showed a higher mean melting temperature of 80.80 ± 0.27 °C, with a statistically significant difference between the two groups (p < 0.01). The assay demonstrated high specificity with no secondary melting peaks or nonspecific amplification products. The clear thermal separation between the two pathogens confirms the reliability of melting curve analysis as a rapid molecular discrimination tool. These findings highlight the potential of HRM-based real-time PCR as a rapid screening approach for foodborne pathogens in poultry products and support its application in routine food safety monitoring and epidemiological surveillance programs. This study represents one of the first applications of HRM-based molecular differentiation of foodborne pathogens isolated from poultry in Iraq.