Development and evaluation of a PCR-microplate capture hybridization method for direct detection of verotoxigenic Escherichia coli strains in artificially contaminated food samples

For the purpose of detecting, directly in food, veroxigenic Escherichia col i, a microplate hybridization method fur the detection of PCR products from the SLT I and SLT II genes, was developed and evaluated. Two pairs of prim ers and two probes, specific for the SLT I gene and for the SLT II gene, we re designed and tested. For the strains containing both genes, two PCR prod ucts of different molecular weights were obtained, whereas when only one ge ne was present only one fragment resulted from PCR. The use of the biotin-l abeled probes allowed the immobilization of the PCR products in the microti ter plate wells and by this means their detection was possible using an ELI SA-based technique. Forty artificially contaminated and fifty naturally con taminated food samples were analyzed by using the PCR-microplate hybridizat ion technique developed in this study. All the artificially contaminated fo od samples were positive, independently of the number of cells inoculated b efore the enrichment step, whereas the naturally contaminated food samples were all negative. (C) 2000 Elsevier Science B.V. All rights reserved.