Detection of Escherichia coli and identification of enterotoxigenic strains by primer-directed enzymatic amplification of specific DNA sequences

Abstract

The polymerase chain reaction (PCR) was used to amplify DNA sequences from the malB operon of Escherichia coli. All E. coli strains tested yielded the specific DNA fragment. No amplification products were obtained with other Enterobacteriaceae. E. coli strains which produce enterotoxins were identified with additional primer pairs specific for the genes coding for the heat-labile toxin type I (LTI) and the heat-stable toxin type I (STI). Amplification products were identified by DNA-DNA hybridization. Alternatively, restriction endonuclease analysis was used for identification and to distinguish between different alleles of the enterotoxin genes. The detection limit was 10 bacteria. The PCR systems were validated by testing 27 E. coli of known enterotoxigenic properties. The PCR results were consistent with factual toxin production as determined by immunoassays. In addition, 58 E. coli strains isolated from soft cheese and mayonnaise were analyzed by PCR. One strain from a cheese sample was found to have the genetic information for STI production. This strain produced STI as determined by enzyme-linked immunosorbent assay.