Simultaneous Enrichment of Salmonella spp, Escherichia coli O157:H7, Vibrio parahaemolyticus, Staphylococcus aureus, Bacillus cereus, and Listeria monocytogenes by Single Broth and Screening of the Pathogens by Multiplex Real-time PCR

Abstract

Simultaneous enrichment broth (SEB) was developed for the single-enrichment simultaneous screening of six major food poisoning bacteria. After enrichment in SEB for 18 h at 37°C, viable counts of six major food poisoning bacteria (Staphylococcus aureus, Salmonella spp., Escherichia coli O157:H7, Vibrio parahaemolyticus, Bacillus cereus, and Listeria monocytogenes) were sufficient for 5' nuclease multiplex real-time PCR assay using existing primers and probes. By labeling the probes with three different fluorescent dyes, the assay could be carried out in 2 tubes. The whole process, including enrichment and PCR, was completed within 24 h and the detection limits for the target bacteria from the food sample (boiled chicken) were 36 cfu/25 g for S. aureus, 5.3 cfu/25 g for Salmonella spp, 2.9 cfu/25 g for E. coli O157:H7, 2.0 cfu/25 g for V. parahaemolyticus, 5.5 cfu/25 g for B. cereus, and 6.2 cfu/25 g for L. monocytogenes. The results were comparable to conventional methods that require 4-6 days.