Purpose: The purpose of this work was to develop a bead suspension Flow Cytometry Immunoassay for the simultaneous detection of E. coli serotypes O157:H7 and O26, and 5 serovars of Salmonella enterica enterica (S. Enteritidis, S. Typhimurium, S. Virchow, S. Newport and S. Meleagridis).
Methods: Polyclonal antibodies were produced against specific strains of E. coli O157:H7 and E. coli O26, whereas a generic polyclonal antibody was generated to cover the 5 Salmonella serovars. Individual bead sandwich assays were developed, and then combined in a triplex bead- test to simultaneously detect E. coli O157:H7, E. coli O26, and the 5 O-groups of Salmonella.
Results: The multiplex assay presented a Limit of Detection in buffer of 104-105 CFU mL–1. No additional cross-reactivity was found with other serotypes of E. coli, Salmonella serovars, or additional Enterobacteria, with the exception of the test O157:H7 which showed positive signal with S. Urbana [shared antigen O:30 (N)], and the test Salmonella which recognized S. Montevideo [shared antigen O:7 (C1) with S. Virchow]. Different enrichment broths were evaluated to determine the recovery of foodborne pathogens in artificially contaminated meat samples (beef and poultry). Matrix interference, influence of competitive flora, and sensitivity for all the media have been compared. Two broths have shown higher growth rates and better recoveries.
Significance: These results indicate that bead-based Flow Cytometry Immunoassays are suitable for the simultaneous, rapid (less than two hours) and high-throughput detection of pathogenic Enterobacteria in food commodities. Further work is ongoing to expand the scope of the test to other significant E. coli serotypes and Salmonella spp.