Detection of enterotoxigenic Escherichia coli in faecal specimens by acetylaminofluorene-labelled DNA probes.
The present study describes acetylaminofluorene(AAF)-modified DNA probes for the identification of heat-labile (LT) and heat-stable (porcine STp and human STh) toxins from enterotoxigenic Escherichia coli (ETEC). AAF probes were compared with established biotinylated probes and bioassays. Ultracentrifugation was not necessary in the preparation of AAF-labelled probes, and the procedures, i.e. chemical modification of probes, hybridization and immunodetection steps, were optimized to detect ETEC by colony hybridization or direct dot blot techniques. The method combines chemical labelling (covalent attachment of AAF group to guanosine is achieved by treatment of DNA with N-acetoxy-2-acetylaminofluorene) and detection of the hybridized target DNA by means of anti-AAF monoclonal antibodies and alkaline phosphatase-labelled second antibodies.
Begaud
E
E
Institut Pasteur de Nouvelle Calédonie, Laboratoire des Bactéries Entéropathogènes, Nouméa.
Germani
Y
Y
D015073:2-Acetylaminofluorene; D000293:Adolescent; D001710:Biotin; D002648:Child; D002675:Child, Preschool; D015342:DNA Probes; D004269:DNA, Bacterial / Q000235:genetics; D003967:Diarrhea / Q000382:microbiology; D004768:Enterotoxins / Q000032:analysis*; D004926:Escherichia coli / Q000737:chemistry* / Q000302:isolation & purification; D004927:Escherichia coli Infections / Q000175:diagnosis / Q000382:microbiology; D005243:Feces / Q000382:microbiology*; D006801:Humans; D015151:Immunoblotting; D066298:In Vitro Techniques; D007223:Infant; D007231:Infant, Newborn; D009693:Nucleic Acid Hybridization
D015342:DNA Probes; D004269:DNA, Bacterial; D004768:Enterotoxins; D001710:Biotin; D015073:2-Acetylaminofluorene