THE USE OF TIME-RESOLVED FLUOROIMMUNOASSAY TO SIMULTANEOUSLY DETECT ESCHERICHIA COLI O157:H7, SALMONELLA ENTERICA SEROVAR TYPHIMURIUM AND SALMONELLA ENTERICA SEROVAR ENTERIDITIS IN FOODS1

Immunomagnetic beads (IMB) were used to capture and concentrate Escherichia coli O157:H7, Salmonella enterica serovar Typhimurium and Salmonella enterica serovar Enteriditis. The captured bacteria were allowed to form sandwiched complexes with europium (Eu) labeled anti-E. coli O157 antibodies and/or samarium (Sm) labeled anti-Salmonella antibodies. After washing the complexes utilizing a magnetic particle concentrator (MPC), the lanthanide labels were extracted from the cell/antibody complexes utilizing PerkinElmer Wallac Enhancement Solution (PerkinElmer Wallac, Turku, Finland) which causes the release of the Eu or Sm from the antibodies and the formation of highly fluorescent Eu-(2-NTA)3(TOPO)2–3 or Sm-(2-NTA)3(TOPO)2,3, micellar complexes. The specific time-resolved fluorescence (TRF) associated with Eu and Sm were measured to estimate the quantity of E. coli and Salmonella, respectively. The sensitivity of the method was in the range of 103 to 104 CFU/mL for pure cultures of E. coli O157:H7 and Salmonella enterica serovar Typhimurium or Salmonella enterica serovar Enteriditis. The approach of applying sandwich complexes yielded a minimum cross-reactivity with non-targeted bacteria. After an enrichment of spiked hamburger, turkey or pork for 4.5 h at 37C in EC medium containing novobiocin, one CFU/g of either E. coli or Salmonella or both was detected. The results demonstrated that low levels of E. coli O157:H7, Salmonella enterica serovar Typhimurium and Salmonella enterica serovar Enteriditis in foods could be detected alone or in-pair by the TRF approach within 8h.