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 Escherichia 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 of 10(2) CFU per PCR (10 microliters) was necessary to amplify E. coli O157:H7-specific bands by multiplex PCR. Food particles as well as various unknown metabolic by-products of bacteria inhibited the PCR, but a simple two-step filtration procedure eliminated this inhibition. To reliably generate PCR products, an E. coli inoculum of 10(3) CFU g of food slurry-1 in a nonspecific medium was required with 6 h of enrichment at 37 degrees C. However, when the food homogenate was incubated overnight, E. coli O157:H7 at an initial inoculum of even 1 CFU g-1 was detected.