Detection of Campylobacter pylori DNA by hybridisation with non-radioactive probes in comparison with a 32P-labelled probe.
A dot-blot hybridisation assay for the detection of Campylobacter pylori was used to compare a 32P-labelled probe with two biotinylated probes and a sulphonated probe. The minimum amount of pure C. pylori DNA that could be detected by the 32P-labelled probe was 100 pg, which corresponded to 5 x 10(4) bacteria. A biotin-labelled DNA (biotin-DNA) probe together with the BluGeneTM detection system produced by Bethesda Research Laboratories (BRL), and a sulphonated probe and ChemiprobeTM detection system (Orgenics) gave similar levels of sensitivity; nylon membranes could be used with both these non-radioactive detection systems. However, a photobiotin-labelled DNA (photobiotin-DNA) probe and detection system produced by Biotechnology Research Enterprises S.A. (BRESA) gave optimum results only with nitrocellulose membranes, and was quantitatively 100 times less sensitive than the other types of probe. The detection systems for the biotin-DNA and photobiotin-DNA probes produced non-specific reactions with crude bacterial blots of heterologous organisms; these non-specific reactions could be removed by treating the dot blots with proteinase K, but not by treatment with RNAase. The sulphonated probe and detection system did not give any reaction with heterologous organism blots.
Wetherall
B L
BL
Department of Clinical Microbiology, Flinders Medical Centre, Bedford Park, Australia.
McDonald
P J
PJ
Johnson
A M
AM
D001386:Azides; D001710:Biotin / Q000031:analogs & derivatives; D002167:Campylobacter / Q000235:genetics*; D004269:DNA, Bacterial / Q000032:analysis*; D008567:Membranes, Artificial; D009693:Nucleic Acid Hybridization; D009757:Nylons; D010761:Phosphorus Radioisotopes; D011933:Reagent Kits, Diagnostic; D013451:Sulfonic Acids
D001386:Azides; D004269:DNA, Bacterial; D008567:Membranes, Artificial; D009757:Nylons; D010761:Phosphorus Radioisotopes; D011933:Reagent Kits, Diagnostic; D013451:Sulfonic Acids; D001710:Biotin; C045549:photobiotin