Polymerase chain reaction for detection of Escherichia coli strains producing cytotoxic necrotizing factor type 1 and type 2 (CNF1 and CNF2)

Abstract

Cytotoxic necrotizing factors type 1 (CNF1) and type 2 (CNF2) are dermonecrotic protein toxins produced by human and animal clinical isolates of Escherichia coli. In this study, two pairs of oligonucleotide primers were designed to amplify fragments of the genes for CNF1 and CNF2 by the polymerase chain reaction (PCR). We also described a further primer pair which amplify fragments of both CNF1 and CNF2 genes. Amplification products of the expected size (543-bp for CNF1, 543-bp for CNF2 and 533-bp for CNFs) were detected in all 240 necrotoxigenic Escherichia coli (NTEC) strains tested. In contrast, no amplification products were seen when the PCR reaction was performed with DNA obtained from 69 non-NTEC bacterial strains (including 13 enterotoxigenic, 11 verotoxigenic and 45 non-toxigenic strains). Furthermore, the toxin genotypes determined by PCR corresponded to the phenotypic results of HeLa cell assay. Therefore, the specificity and sensitivity for the three pairs of oligonucleotide primers designed in this study was 100%. The PCR protocol describe here permits rapid and accurate detection of CNF1 and CNF2 genes in Escherichia coli strains isolated from clinical material and contaminated foods.