Detection of enterotoxigenic Escherichia coli in stool samples by using nonradioactively labeled oligonucleotide DNA probes and PCR

Abstract

The detection of heat-labile enterotoxin LT-A and heat-stable enterotoxin ST Ia and ST Ib genes from enterotoxigenic Escherichia coli (ETEC) by using oligonucleotide DNA probes and the PCR was evaluated in reconstruction experiments and by testing stool specimens from 29 healthy subjects and from 50 patients with diarrhea who had returned from the (sub)tropics. ETEC strains were detected in concentrations ranging from 10(6) to 10(8) CFU/g of feces when oligonucleotide probes were applied to colony blots from five randomly picked E. coli-like colonies from CLED (cystine lactose electrolyte deficient) agar plates inoculated with the feces. When these probes were applied to blots from whole stool cultures collected from the agar plates (sweep blot), the detection limit was 10(6) CFU/g of feces. PCR of the sweep material could detect toxin genes when the concentration of ETEC strains was 10(2) CFU/g of feces. Results obtained with stool specimens from 29 healthy control subjects were negative. Testing stool specimens from 50 patients confirmed the observation that the number of samples containing ETEC enterotoxin genes was higher when PCR of sweeps was used than when oligonucleotide DNA probe hybridization of either sweep blots or colony blots was used. Furthermore, PCR of sweeps is an easy and rapid method which does not require DNA extraction and purification from fecal specimens.