DEVELOPMENT OF A COLONY LIFT IMMUNOASSAY TO FACILITATE RAPID DETECTION AND QUANTIFICATION OF ESCHERICHIA-COLI 0157-H7 FROM AGAR PLATES AND FILTER MONITOR MEMBRANES

E. coli O157:H7 is a food-borne adulterant that can cause hemorrhagic ulcerative colitis and hemolytic uremic syndrome. Faced with an increa sing risk of foods contaminated with E. coli O157:H7, food safety offi cials are seeking improved methods to detect and isolate E. coli O157: H7 in hazard analysis and critical control point systems in meat- and poultry-processing plants. A colony lift immunoassay was developed to facilitate the positive identification and quantification off. coli O1 57:H7 by incorporating a simple colony lift enzyme-linked immunosorben t assay with filter monitors and traditional culture methods. Polyviny lidene difluoride (PVDF) membranes (Millipore, Bedford, Mass.) were pr ewet with methanol and were used to make replicates of every bacterial colony on agar plates or filter monitor membranes that were then rein cubated for 15 to 18 h at 36 +/- 1 degrees C, during which the colonie s not only remained viable but were reestablished. The membranes were dried, blocked with blocking buffer (Kirkegaard and Ferry Laboratories [KPL], Gaithersburg, Md.), and exposed for 7 min to an affinity-purif ied horseradish peroxidase-labeled goat anti-E. call O157 antibody (KF L). The membranes were washed, exposed to a 3,3',5,5'-tetramethylbenzi dine membrane substrate (TMB; KFL) or aminoethyl carbazole (AEC; Sigma Chemical Co., St. Louis, Mo.), rinsed in deionized water, and air dri ed. Colonies of E. coli O157:H7 were identified by either a blue (via TMB) or a red (via AEC) color reaction. The colored spots on the PVDF lift membrane were then matched to their respective parent colonies on the agar plates or filter monitor membranes. The colony lift immunoas say was tested with a wide range of genera in the family Enterobacteri aceae as well as different serotypes within the E. coli genus. The col ony lift immunoassay provided a simple, rapid, and accurate method for confirming the presence off. call O157:H7 colonies isolated on filter monitors or spread plates by traditional culture methods. An advantag e of using the colony lift immunoassay is the ability to test every co lony serologically on an agar plate or filter monitor membrane simulta neously for the presence of the E. coli O157 antigen. This colony lift immunoassay has recently been successfully incorporated into a rapid- detection, isolation, and quantification system for E. coli O157:H7, d eveloped in our laboratories for retail meat sampling.