title: Time-resolved fluorescence immunoassay (TRFIA) for the detection of Escherichia coli O157:H7 in apple cider.
abstract: An immunoassay based on immunomagnetic separation and time-resolved fluorometry was developed for the detection of E. coli O157:H7 in apple cider. The time-resolved fluorescent immunoassay (TRFIA) uses a polyclonal antibody bound to immunomagnetic beads as the capture antibody and the same antibody labeled with europium as the detection antibody. Cell suspensions of 10(1) to 10(8) E. coli O157:H7 and K-12 organisms per ml were used to test the sensitivity and specificity of the assay. The sensitivity of the assay was 10(3) E. coli O157:H7 cells with no cross-reaction with K-12. Pure cultures of E. coli O157:H7 (10(1) to 10(5) CFU/ml) in apple cider could be detected within 6 h, including 4 h for incubation in modified EC broth with novobiocin and 2 h for the immunoassay. When apple cider was spiked with 1 to 10(3) CFU/ml of E. coli O157:H7 and 10(6) CFU/ml of K-12, our data show that the high level of K-12 in apple cider did not impede the detection of low levels of O157:H7. The minimum detectable numbers of cells present in the initial inoculum were 10(2) and 10(1) CFU/ml after 4- and 6-h enrichment. The TRFIA provides a rapid and sensitive means of detecting E. coli O157:H7 in apple cider.
authors.affiliation: U.S. Department of Agriculture, Agricultural Research Service, Eastern Regional Research Center, 600 E. Mermaid Lane, Wyndmoor, PA 19038, USA. lyu@arserrc.gov
authors.forename: L S L
authors.initials: LS
authors.lastname: Yu
authors.forename: S A
authors.initials: SA
authors.lastname: Reed
authors.forename: M H
authors.initials: MH
authors.lastname: Golden
mesh_terms: D000907:Antibodies, Bacterial / Q000276:immunology / Q000378:metabolism; D001628:Beverages / Q000382:microbiology*; D015169:Colony Count, Microbial; D019453:Escherichia coli O157 / Q000254:growth & development / Q000276:immunology / Q000302:isolation & purification*; D005063:Europium / Q000378:metabolism; D007118:Immunoassay / Q000379:methods*; D018189:Immunomagnetic Separation; D027845:Malus / Q000382:microbiology*; D012680:Sensitivity and Specificity; D013997:Time Factors
chemical_list: D000907:Antibodies, Bacterial; D005063:Europium