Development of multiplex PCR for detection of foodborne pathogens in fresh produce

Abstract Foodborne pathogens present a significant public health concern where fresh produce is a key agricultural product. Rapid and sensitive detection methods are essential to ensure the safety of such produce. This study aimed to develop and optimize a modified multiplex PCR (mPCR) assay, which incorporates enhancements to conventional PCR, for the simultaneous detection of Escherichia coli, Salmonella, and Listeria monocytogenes in fresh produce. The specificity of each primer pair was validated using 15 strains, confirming 100% accurate detection of pathogenic strains without cross-reactivity. Since no false positives were observed, the assay demonstrated 100% precision, highlighting its reliability in distinguishing target pathogens. The sensitivity of the mPCR assay was demonstrated through serial dilutions, detecting Salmonella down to 10 fg µl⁻1, L. monocytogenes to 100 fg µl⁻1, and E. coli to 1 pg µl⁻1. The mPCR assay was then successfully applied to romaine lettuce and kale, demonstrating its effectiveness in detecting pathogens in mixed samples inoculated at varying concentrations (109 to 101 CFU ml⁻1). Kale exhibited greater sensitivity, detecting pathogens at lower levels, while romaine lettuce also provided consistent detection. This study highlights the potential of mPCR for enhancing food safety by providing rapid and sensitive pathogen detection in fresh produce.