Simultaneous Detection of Four Foodborne Pathogens in Raw Freshwater Fish Using High-Resolution Melting Analysis

Many countries around the world feature raw fish in their cuisine, which is valued for its unique flavor. However, raw fish may be easily contaminated with foodborne pathogens including <i>Listeria monocytogenes</i>, <i>Salmonella</i>, <i>Vibrio parahaemolyticus</i>, and <i>Staphylococcus aureus</i>. Herein, a method was established that integrated a multiplex polymerase chain reaction (PCR) and high-resolution melting (HRM) curve assay for the simultaneous detection of these four foodborne pathogens. The target genes of the bacteria were amplified by PCR and subsequently analyzed using HRM. Differentiation was achieved based on the melting temperature (Tm) values of their respective amplicons. The detection limit of the PCR-HRM assay was 0.02-0.1 ng/µL. In addition, the Tm remained nearly constant across various concentrations of genomic DNA derived from the target bacteria. The assay demonstrated perfect specificity (8/8) and a sensitivity of 5/5 for <i>L. monocytogenes</i>, 2/2 for <i>Salmonella</i>, 3/3 for <i>V. parahaemolyticus</i>, and 3/3 for <i>S. aureus</i>. No significant interference occurred when genomic DNA from the four target bacteria was co-extracted with DNA from eight non-target strains. Furthermore, the assay offers advantages including operational simplicity, high efficiency, accurate results, reduced detection time, and lower costs, rendering it well-suited for food safety applications in the aquatic products processing industry.