POLYMERASE CHAIN-REACTION FOR DETECTION OF ESCHERICHIA-COLI STRAINS PRODUCING CYTOTOXIC NECROTIZING FACTOR TYPE-1 AND TYPE-2 (CNF1 AND CNF2)

Cytotoxic necrotizing factors type 1 (CNF1) and type 2 (CNF2) are derm onecrotic protein toxins produced by human and animal clinical isolate s of Escherichia coli. In this study, two pairs of oligonucleotide pri mers were designed to amplify fragments of the genes for CNF1 and CNF2 by the polymerase chain reaction (PCR). We also described a further p rimer pair which amplify fragments of both CNF1 and CNF2 genes. Amplif ication products of the expected size (543-bp for CNF1, 543-bp for CNF 2 and 533-bp for CNFs) were detected in all 240 necrotoxigenic Escheri chia coli (NTEC) strains tested. In contrast, no amplification product s were seen when the PCR reaction was performed bacterial strains (inc luding 13 enterotoxigenic, 11 verotoxigenic and 45 non-toxigenic strai ns). Furthermore, the toxin genotypes determined by PCR corresponded t o the phenotypic results of HeLa cell assay. Therefore, the specificit y and sensitivity for the three pairs of oligonucleotide primers desig ned in this study was 100%. The PCR protocol describe here permits rap id and accurate detection of CNF1 and CNF2 genes in Escherichia coli s trains isolated from clinical material and contaminated foods.