A new single-tube platform of melting temperature curve analysis based on multiplex real-time PCR using EvaGreen for simultaneous screening detection of Shiga toxin-producing Escherichia coli, Salmonella spp. and Listeria monocytogenes in food

Abstract

Shiga toxin-producing Escherichia coli (STEC), Salmonella spp. and Listeria monocytogenes are continuously reported as causative agents of great concern regarding food safety and widespread contamination in many food varieties. Therefore, their simultaneous detection may be advantageous in terms of cost, time and labor savings and simplicity. This study developed a new, simple platform of multiplex real-time polymerase chain reaction (mRT-PCR) for specific, sensitive and rapid detection of STEC, Salmonella spp. and L. monocytogenes in food. The single-tube mRT-PCR format was developed by combining an 18 h enrichment step in simultaneous enrichment broth, boiling based on DNA extraction assay and a mRT-PCR detection system based on melting curve analysis using a fluorescent dye (EvaGreen) for detection of the presence or absence of the three target bacterial pathogens in food samples. Three specific peaks were clearly detected with average melting temperatures of 84.52 ± 0.90 °C, 87.51 ± 0.54 °C and 79.32 °C ± 0.48 °C for STEC, Salmonella spp. and L. monocytogenes, respectively. The sensitivity and specificity of these newly developed mRT-PCR platforms were further investigated using artificially and naturally contaminated food samples. The relative sensitivity, relative specificity and relative accuracy were all 100%, with a detection limit of 1 cfu for each target pathogen in 25 g of food sample. The developed platform of EvaGreen-based single-tube mRT-PCR for detection of the three target pathogenic bacteria in food samples provided results of absence or presence within 20 h. The newly developed mRT-PCR platform in this study offers a promising approach for simple, rapid, sensitive, specific and accurate detection of the three target bacterial pathogens in food.