Detection of Campylobacter pylori DNA by hybridisation with non-radioactive probes in comparison with a 32P-labelled probe

Abstract

Summary.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 lo4 bacteria. A biotin-labelled DNA (biotin-DNA) probe together with the BluGeneTM detection system produced b Bethesda Research Laboratories (BRL), and a sulphonated probe 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.and ChemiprobeT x detection system (Orgenics) gave similar levels of sensitivity;

Occurrence of aminoglycoside phosphotransferase subclass I and II structural genes among Enterobacteriaceae spp. isolated from meat samples

“…Subsequently, the resistance determinant was cloned and the resultant chimeric plasmid was used to develop a non-radioactive DNA probe. Non-radioactive biotin-labelled (Carter et al 1989;Wetherall et al 1988;Zeph and Stotzky 1989) and acetylaminoflurenelabelled (Chevrier et al 1989) DNA probes have been used to detect specific sequences in bacterial and viral DNA. The sensitivity, safety, long storage life, reusability, and low background hybridization signals have made these probes a convenient alternative to radiolabelled DNA probes.…”

A Review of Diagnostic Techniques for <i>Helicobacter pylori </i>Infection

“…Differ ent methods of antibody detection have been developed, including complement fixation tests [57], passive haemagglutination assays [58], enzyme-linked immunosorbent assays (ELISA) [52,59], and immunoblot techniques [60,61]. Detection o f//, pylori by means ofin situ DNA hybridization is a sophisticated method not yet available to most investiga tors [62], Currently, the IgG ELISA is the most common technique. In the first-genera tion assays, cross-reactivity with Campylo bacter jejuni was found to be a confounding factor in the interpretation of the results [63].…”

Use of a capacitive affinity biosensor for sensitive and selective detection and quantification of DNA—A model study

“…Determination of specific DNA-sequences in clinical or food samples can result in the detection and identification of certain infectious organisms [1]. Various DNA-sensors with labeled probes have been reported; where the use of radioisotope-labeled ( 125 I or 132 P) DNA-probes have been reported frequently [2], [3], [4]. However, apart from high sensitivities, the use of isotope-labeled reagents is restricted because of the potential danger of radioactivity.…”