DETECTION OF ESCHERICHIA COLI O157:H7 THROUGH THE FORMATION OF SANDWICHED COMPLEXES WITH IMMUNOMAGNETIC AND FLUORESCENT BEADS†

Abstract

ABSTRACT A new fluorescent sandwich method for the detection of Escherichia coli O157:H7 in ground beef was developed. Immunomagnetic beads (IMBs) precoated with anti‐E. coli O157:H7 antibody were used to capture and concentrate E. coli O157:H7 present in ground beef. Streptavidin‐coated fluorescent beads with anti‐E. coli O157:H7 to form immunofluorescent beads (IFBs) were used to detect the E. coli O157:H7 captured by the IMBs by forming IMBM–E. coli O157:H7N–IFBO sandwich complexes, where the subscripts M, N and O were variable but integral numbers. This sandwich technique was able to detect 500 cfu of the bacteria in a 0.5‐mL sample taken from a quantified and diluted broth culture. The method was then applied to detect E. coli O157:H7 in artificially contaminated ground beef. Known quantities of freshly cultured E. coli O157:H7 cells were added to 25‐g ground beef patties held in sterile bags with filters. The inoculated patties were stored at 7C overnight; 75 mL of E. coli broth was added to each sample, and the bags were incubated at 40C with shaking at 160 rpm for 4.5 h. After the enrichment, E. coli O157:H7 in the filtrate was captured and concentrated with IMBs and further conjugated with IFBs for detection. The results demonstrate that, after a 4.5‐h enrichment period, the developed procedure can detect the presence of E. coli O157:H7 in ground beef previously spiked with 1 cfu/g.