Patent ID: 6268143
Filing Date: 2001-07-31
Classification: C12Q

Abstract:
A method of determining whether a sample is free of the presence of E. coli O157:H7 using an eaeA-based 5' nuclease assay, wherein SZ primers are used as a nuclease target in said assay, comprising the steps of:collecting a sample to be tested for the possible presence of E. coli O157:H7;preparing an enrichment broth culture of said sample;extracting DNA from said culture;amplifying said DNA by PCR using E. coli O157:H7 eaeA-specific primers, wherein said primers amplify all or part of a sequence corresponding to nucleotides 28 to 659 of GenBank accession number U32312 in the presence of a fluorogenic probe specific for an E. coli O157:H7 eaeA-specific sequence positioned between said primers, wherein said fluorogenic probe is selected from the group consisting of SZI-97 having SEQ ID NO: 3 and SZI-107 having SEQ ID NO-4; andanalyzing the fluorescence of said sample, wherein an increase in fluorescence indicates the possible presence of E. coli O157:H7 in said sample, and no increase in fluorescence indicates that the sample is free of E. coli O157:H7.