Simultaneous enumeration of Campylobacter jejuni and Salmonella enterica genome equivalents by melting curve analysis following duplex real time PCR in the presence of SYBR Green

Chicken meat and eggs contaminated with Salmonella enterica and Campylobacter jejuni are among the major causes of gastrointestinal infections in humans. Determining the numbers of these pathogens at various stages of the food supply chain is critical to the validation of steps designed to produce safer food. In the current study, duplex real time PCR in the presence of SYBR Green was carried out with DNA extracted from pure cultures of the two pathogens and from chicken meat samples spiked with them. The peak areas of derivative of dissociation curves (PADDC), obtained after 35 PCR cycles were calculated and plotted against known genome equivalents (GEs) in a standard curve. The method provided an estimation for the number of GEs in a 25 μL PCR sample when 102-105GEs were present, similar to those obtained with duplex qPCR based on TaqMan probes by other authors, but with reduced costs.