Development and evaluation of a multiplex real-time PCR (qPCR) assay targeting ttrRSBCA locus and invA gene for accurate detection of Salmonella spp. in fresh produce and eggs

Contamination of foods, especially produce and eggs, with Salmonella spp. is a major concern for public health. Therefore the development of a rapid method for Salmonella detection in those two important food commodities is urgently needed. The main objective of our study was to develop and evaluate a multiplex TaqMan-based real time PCR assay for detection of Salmonella spp. targeting invA gene and ttrRSBCA locus. The detection limit of this assay, 13 copies of genomic invA gene and ttrRSBCA locus, was determined by 10-fold dilutions of DNA from S. Typhimurium (strain SARA9). Inclusivity and exclusivity of the assay were assessed using 101 Salmonella spp. (including all Salmonella species and subspecies) and 48 non-S. enterica strains, respectively. Samples of cilantro (coriander leaves), lettuce, parsley, spinach, tomatoes and jalapeno peppers were seeded with four different Salmonella serovars at levels of 1-20 (low level) and 105 (high level) CFU/25 g. The inoculated samples were assayed according to the FDA Salmonella culture method in the Bacteriological Analytical Manual (BAM). Eggs were seeded with two different S. Enteritidis strains at the level used for the produce samples and were assayed according to the USDA Laboratory Guidebook Salmonella culture method. The contaminated samples were analyzed with the multiplex TaqMan-based assay developed in this study. Comparable results were obtained by the qPCR and the two bacteriological methods. Levels as low as 2 CFU/25 g for produce were detected with the qPCR method according to the BAM and 5 CFU/100 g for eggs were detected with the qPCR method according to the USDA Laboratory Guidebook. False negatives (inhibition of PCR reaction) were ruled out through the use of a DNA internal amplification control (IAC). The qPCR multiplex assay developed in this study allows rapid and accurate detection of Salmonella spp. in six high-risk produce commodities and eggs, and has the potential to be used with other food matrices.

► A real time PCR assay for detection of Salmonella using two targets was evaluated. ► Artificial contaminated samples were analyzed by microbiology methods and the qPCR. ► Both methods provided comparable results. ► Levels as low as 2 CFU/25 g for produce and 5 CFU/100 g for eggs were detected. ► The qPCR assay developed here allowed rapid and accurate detection of Salmonella.