Characterization of an rRNA gene-specific cDNA probe: applications in bacterial identification

Abstract

Summary: Discontinuous DNA complementary to Escherichia coli 16S + 23S ribosomal RNA was synthesized by random oligonucleotide priming using reverse transcriptase. cDNA generated from native or denatured rRNA template was labelled by incorporation of either [α-32P]dCTP or digoxigenin-labelled dUTP during synthesis, followed by template hydrolysis. The specific activity of the radiolabelled cDNA was 107-108 c.p.m. (μg rRNA template)−1 with 60-92% incorporation after 5 h. The length of the reverse transcript was between 20 and 1140 nucleotides and was unaffected by exclusion of primer. The cDNA probe could detect 3 pg rRNA by quantitative slot blot. In the non-radiolabelling digoxigenin system 3 μg template gave 0·5-2·0 μg cDNA after 24 h with a length of between 100 and 1225 bases. This probe could detect 50 pg rRNA. Probes were evaluated in the comparison of Pasteurella haemolytica biotypes by hybridization to Southern blots of restriction-endonuclease-digested total DNA. The digoxigenin-labelled probe was used to identify clinical isolates of Campylobacter jejuni to demonstrate its potential use in laboratories requiring high-sensitivity detection without the use of radioisotopes.