A SIMPLE FILTRATION TECHNIQUE TO DETECT ENTEROHEMORRHAGIC ESCHERICHIA-COLI O157-H7 AND ITS TOXINS IN BEEF BY MULTIPLEX PCR

Primers, specific for a unique base substitution in uidA of Escherichi a coli O157:H7, were coupled with oligonucleotides for the shiga-like toxin I (SLT-I) and SLT-II genes in a multiplex PCR assay, A minimum o f 10(2) CFU per PCR (10 mu l) was necessary to amplify E. coli O157:H7 -specific bands by multiplex PCR. Food particles as well as various un known metabolic by-products of bacteria inhibited the PCR, but a simpl e two-step filtration procedure eliminated this inhibition. To reliabl y generate PCR products, an E. coli inoculum of 10(3) CFU g of food sl urry(-1) in a nonspecific medium was required with 6 h of enrichment a t 37 degrees C. However, when the food homogenate was incubated overni ght, E. coli O157:H7 at an initial inoculum of even 1 CFU g(-1) was de tected.