Synthesis of a biotinylated DNA probe to detect ribosomal RNA cistrons in Providencia stuartii

• Published in: FEMS microbiology letters 1987-12, Vol.48 (1‐2), p.283-287
• Main Authors: Pitcher, D.G., Owen, R.J., Dyal, P., Beck, A.
• Format: Article
• Language: English
• Subjects: Bacteriological methods and techniques used in bacteriology; Bacteriology; Biological and medical sciences; Biotin labelling; cDNA synthesis; Fundamental and applied biological sciences. Psychology; Microbiology; Providencia stuartii; Ribosomal RNA cistrons
• ISSN: 0378-1097; 1574-6968
• DOI: 10.1111/j.1574-6968.1987.tb02557.x

A simple and convenient procedure that obviates the need for DNA cloning and radiolabelling is described for producing a rDNA hybridisation probe for use in bacterial strain characterisation. Bulk rRNA, comprising a mixture of 16S and 23S components, was isolated from Providencia stuartii NCTC11800 and purified by CsC1‐cushion ultracentrifugation. The pure RNA was used as a template for synthesis of a single‐strand copy cDNA using reverse transcriptase and random primers, with simultaneous biotin‐11‐dUTP labelling. EcoRI digests of chromosomal DNA from the type strain and a sample of 10 clinical isolates of P. stuartii were examined by Southern blot hybridisation on nylon membranes using the cDNA probe. All 11 strains had similar but not identical hybridisation patterns of 5–7 bands (detected colorimetrically) with sizes of about 5–19 kb. Our results indicated that the probe provided a sensitive means of detecting rRNA cistrons in P. stuartii. The method should be applicable to any group of microorganisms.