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.
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D000469:Alkaline Phosphatase; D001315:Australia; D001427:Bacterial Toxins / Q000032:analysis* / Q000235:genetics; D003967:Diarrhea / Q000382:microbiology; D004768:Enterotoxins / Q000032:analysis* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000302:isolation & purification*; D029968:Escherichia coli Proteins; D005006:Ethnicity; D005243:Feces / Q000382:microbiology*; D006801:Humans; D009693:Nucleic Acid Hybridization; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis
D001427:Bacterial Toxins; D004768:Enterotoxins; D029968:Escherichia coli Proteins; D009838:Oligodeoxyribonucleotides; C034151:heat stable toxin (E coli); D000469:Alkaline Phosphatase