1887

Abstract

Discontinuous DNA complementary to 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-P]dCTP or digoxigenin-labelled dUTP during synthesis, followed by template hydrolysis. The specific activity of the radiolabelled cDNA was 10–10 c.p.m. (g rRNA template) 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 μg rRNA by quantitative slot blot. In the non-radiolabelling digoxigenin system 3 g 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 biotypes by hybridization to Southern blots of restriction-endonuclease-digested total DNA. The digoxigenin-labelled probe was used to identify clinical isolates of to demonstrate its potential use in laboratories requiring high-sensitivity detection without the use of radioisotopes.

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1990-08-01
2021-10-24
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