Enzyme-linked synthetic oligonucleotide probes: non-radioactive detection of enterotoxigenic Escherichia coli in faecal specimens

Synthetic oligonucleotides, complementary to unique sequences in the heat stable enterotoxin gene of Escherichia coli specific for humans, were prepared with a 30-atom spacer arm and a 3' terminal sulfhydryl group which was coupled to bromoacetyl-derivatized alkaline phosphatase. The resulting direct enzyme-linked oligonucleotide probes, containing one enzyme molecule per oligonucleotide, successfully diagnosed enterotoxigenic Escherichia coli in clinical specimens by using a modified colony hybridization method and a colorimetric assay. The procedure is rapid, simple and reliable with a sensitivity equivalent to that using 5'-terminally labelled [32p]-oligonucleotide probes. The results indicate that the enzyme-labelled oligonucleotide probes should be applicable to the routine diagnosis of enterotoxigenic Escherichia coli and possess the potential for the detection of other microbial pathogens.