Detection of enterotoxigenic Escherichia coli by dot blot hybridization with biotinylated DNA probes

Abstract

A dot blot hybridization test was developed for the detection of enterotoxigenic E. coli without the use of radioisotopes. Three biotin-labeled DNA (Bio-DNA) probes corresponding to structural genes specifying heat-labile and heat-stable enterotoxins of porcine and human origin were prepared by random priming; label incorporation was significantly higher than that obtained from the use of nick translation. Bio-DNA probes were highly specific when reacted with protein- and RNA-free DNA preparations in a dot blot hybridization assay. The Bio-DNA probe, in which 40% of available thymidines were replaced by a biotin-labeled deoxyuridine, readily detected 160 pg of target DNA mixed with 6 micrograms of carrier DNA. The minimum amount of total DNA required for reliable identification of a single-copy enterotoxin gene of porcine origin within a 5,000-kilobase chromosome was found to be approximately 5 micrograms. Complete agreement among the results of Bio-DNA probe hybridization, [32P]DNA hybridization, and biological assay was demonstrated for 15 (100%) of the clinical isolates. This procedure provides a more suitable approach for the diagnosis of enterotoxigenic E. coli infections in clinical settings than the hybridization assay based on 32P-labeled DNA probes.