Use of real-time polymerase chain reaction and molecular beacons for the detection of Escherichia coli O157 : H7

Molecular beacons (MBs) are oligonucleotide probes that fluoresce upon hybr idization. In this paper, we described the development of a real-time PCR a ssay to detect the presence of Escherichia coli O157:H7 using these fluorog enic reporter molecules. MBs were designed to recognize a 26-bp region of t he rfbE gene, coding for an enzyme necessary for O-antigen biosynthesis. Th e specificity of the MB-based PCR assay was evaluated using various enteroh emorrhagic (EHEC) and Shiga-like toxin-producing (STEC) E. coli strains as well as bacteria species that cross-react with the O157 antisera. All E. co li serotype O157 tested was positively identified while all other species, including the closely related O55 were not detected by the assay. Positive detection of E. coli O157:H7 was demonstrated when >10(2) CFU/ml was presen t in the samples. The capability of the assay to detect E. coli O157:H7 in raw milk and apple juice was demonstrated. As few as 1 CFU/ml was detected after 6 h of enrichment. These assays could be carried out entirely in seal ed PCR tubes, enabling rapid and semiautomated detection of E. coli O157:H7 in food and environmental samples. (C) 2001 Academic Press.