Detection of Verotoxin-Producing Escherichia coli O157:H7 by Multiplex Polymerase Chain Reaction

We constructed primers for multiplex polymerase chain reaction (PCR) to detect verotoxin-producing Escherichia coli (VTEC) O157:H7. The multiplex PCR primers were designed from the sequence of the flagellin structural gene of Escherichia coli flagellar type H7 (GenBank under accession number L07388), and from the sequence of the rfbE gene of Escherichia coli O157:H7 (GenBank under accession number S83460). In addition to these primers, we used a primer pair reported by Karch and Meyer (J. Clin, Microbiol. 27: 2751-2757, 1989) to amplify various VT genes from VTEC. All of the examined specimens (18 isolates) of VT-producing E. coli O157:H7 showed a positive result by the multiplex PCR test with the three sets of primers. The sensitivity of detection for VT-producing E. coli O157:H7 was shown to be at least 3,000 cells per PCR tube.     

Amplification of rfbE and fliC Genes by Polymerase Chain Reaction for Identification and Detection of Salmonella Serovar Enteritidis,Dublin and Gallinarum-Pullorum

Polymerase chain reaction (PCR) primers for O9 antigen (rfbE) and phase 1 flagellin antigen (fliC) were designed for the rapid identification and detection of Salmonella serovar Enteritidis and Dublin. The rfbE primer pairs selectively amplified the rfbE region of group O9 Salmonella serovars. The fliC primer pairs amplified the DNAs of g,m and g,p-type flagellar antigen; Salmonella serovar Enteritidis, Dublin, and Essen. However, DNA from flagellar-negative Salmonella serovar Gallinarum-Pullorum was also amplified. The sensitivity of PCR primer pairs was 10 CFU/assay by boiled DNA preparation and 10² CFU/assay by proteinase K-treated DNA preparation.