Simultaneous Detection of Foodborne Pathogens Using a Real-Time PCR Triplex High-Resolution Melt Assay

Abstract

Foodborne pathogens pose risks to populations all over the world. Pathogens can be used as bioterrorism agents, causing an outbreak that affects many individuals through the consumption of a commonly affected food or beverage. A PCR assay can be used to identify pathogens through their unique melting points using a high-resolution melt assay. Assays can be used to detect the bacteria individually or from a mixture using species-specific primers. An assay was developed to detect and identify three pathogens that routinely cause multistate foodborne outbreaks, as documented by the U.S. Centers for Disease Control and Prevention, Campylobacter jejuni (C. jejuni), Escherichia coli (E. coli), and Salmonella enterica (S. enterica), in single bacterium assays and a multiplex. The primers were targeted to specific and unique gene sequences of each pathogen, including cadF, yedN, and hilA, respectively. Each pathogen was identified by its unique melting temperature in single assays: 78.10 ± 0.58 °C for C. jejuni, 81.96 ± 0.42 °C for E. coli, and 87.55 ± 0.37 °C for S. enterica. The multiplex successfully detected and identified all three of the pathogens with the distinctly separated melt peaks. The PCR high-resolution melt assay also proved to be specific, reproducible, fast, and sensitive in experiments.