DETECTION OF VEROTOXIN-PRODUCING ESCHERIC HIA-COLI IN EXPERIMENTALLY INOCULATED SOFT CHEESE BY POLYMERASE CHAIN-REACTION

According to the Council Directive 92/46/EEC for E. coli in cheese mad e from raw milk and thermized milk and in soft cheese made from heat-t reated milk standards on removal from the processing establishment are determined. Whenever the maximum values are exceeded testing must be carried out for the presence of presumptive pathogenic E. coli in such products. In the last years among pathogenic E. coli especially verot oxigenic E. coli (VTEC) have gained significance. The usefulness of po lymerase chain reaction (PCR) for direct detection of VTEC in soft che eses was investigated. In studies with pure cultures 10(2) cfu VTEC co mbined with 10(7) cfu non-VTEC were detectable by PCR followed by dot blot hybridization of PCR products. For the detection of VTEC in food soft cheeses made from raw milk containing natural bacterial counts of 10(2) E. coli/g and more than 10(5) coliforms/g were experimentally i noculated. By filtration and centrifugation bacteria were collected an d inhibitors removed before heating to release the DNA from bacterial cells. Sensitivity and specificity of the reaction were enhanced by an extraction procedure with Chelex(R) 100. Inocula of 10(3) cfu VTEC/g were directly detectable by PCR and dot blot hybridization of PCR prod ucts. The occurrence of unspecific products, however, requires the con trol of all PCR products by dot blot hybridization.