Improved sensitivity of whole-cell hybridization by the combination of horseradish peroxidase-labeled oligonucleotides and tyramide signal amplification.
The substrate fluorescein-tyramide was combined with oligonucleotide probes directly labeled with horseradish peroxidase to improve the sensitivity of in situ hybridization of whole fixed bacterial cells. Flow cytometry and quantitative microscopy of cells hybridized by this technique showed 10- to 20-fold signal amplifications relative to fluorescein-monolabeled probes. The application of the new technique to the detection of natural bacterial communities resulted in very bright signals; however, the number of detected cells was significantly lower than that detected with fluorescently monolabeled, rRNA-targeted oligonucleotide probes.
Schönhuber
W
W
Max-Planck-Institut für Marine Mikrobiologie, Bremen, Germany.
Fuchs
B
B
Juretschko
S
S
Amann
R
R
D004926:Escherichia coli / Q000302:isolation & purification*; D005434:Flow Cytometry / Q000379:methods; D005456:Fluorescent Dyes / Q000378:metabolism*; D006735:Horseradish Peroxidase / Q000378:metabolism*; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D009841:Oligonucleotides / Q000235:genetics*; D012335:RNA, Ribosomal / Q000235:genetics; D012680:Sensitivity and Specificity
D005456:Fluorescent Dyes; D009841:Oligonucleotides; D012335:RNA, Ribosomal; D006735:Horseradish Peroxidase