Open AccessEnzyme-linked synthetic oligonucleotide probes: non-radioactive detection of enterotoxigenicEscherichia coliin faecal specimens
Author(s) -
Peng Li,
Peter P. Medon,
Derek C. Skingle,
J.B.K. Lanser,
Robert H. Symons
Publication year - 1987
Publication title -
nucleic acids research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 9.008
H-Index - 537
eISSN - 1362-4954
pISSN - 0305-1048
DOI - 10.1093/nar/15.13.5275
Subject(s) - biology , escherichia coli , enterotoxigenic escherichia coli , enzyme , oligonucleotide , microbiology and biotechnology , enterobacteriaceae , oligomer restriction , biochemistry , dna , gene , enterotoxin
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.