A novel approach for simultaneous detection of the most common food-borne pathogens by multiplex qPCR

Abstract

Food contaminated with bacterial pathogens is a great threat to human health and food spoilage, having an impact on public health and the food industry. Research in food safety seeks to develop a practical, rapid, and sensitive detection technique for food-borne pathogens. In the past few decades real-time quantitative polymerase chain reaction (qPCR) has been developed, and multiplex qPCR is a preferred feature. Multiplex qPCR enables simultaneous amplification of many targets of interest in one reaction by using more than one pair of primers. In this study we have developed and evaluated a hydrolysis (TaqMan) probe-based system for simultaneous detection of eight of the most common food-borne pathogens in a single step procedure by multiplex qPCR. A multicolor combinational probe coding (MCPC) strategy was utilized that allows multiple fluorophores to label different probes in combinatorial manner. This strategy enabled simultaneous detection, identification, and quantification of targeted genes. The efficiency of the individual qPCR reactions for each target gene had values comparable to those established for multiplex qPCR, with the detection limits of approximately < 10 copies of DNA per reaction. Pathogen load help predict bacteriological quality status in food products and serve to validate the efficiency of procedures to minimize or eliminate their presence, so newly developed multiplex qPCR was quantitative for each pathogen. During sample preparation, a step to concentrate the target organism from a relatively large sample size, remove all potential PCR inhibitors and yield samples in a volume suitable for qPCR was incorporated.

Improving the Efficiency of Viability-qPCR with Lactic Acid Enhancer for the Selective Detection of Live Pathogens in Foods

“…For UHT milk, the standard plating methods require different enrichment media plus long detection/confirmation times (2 days) to target aerobic or anaerobic viable contaminant bacteria. When it comes to analyzing food samples using qPCR, the complexity of food matrices poses several challenges: the presence of PCR inhibitors, variability in food composition, texture, and structure, the presence of enzymes that degrade DNA, and so on [46][47][48][49][50]. In our experiments, the lowest detection limit was log 2 CFU/mL for viable cells or mixtures with log 2 CFU/mL alive and log 2 CFU/mL heat-killed bacteria.…”