PCR-BASED PREPARATION OF 23S RIBOSOMAL-RNA-TARGETED GROUP-SPECIFIC POLYNUCLEOTIDE PROBES

DNA coding for a variable region within domain III of bacterial 23S rR NA was used as the target for group-specific polynucleotide hybridizat ion probes. The corresponding rDNA was amplified in vitro by the PCR t echnique in combination with a pair of primers specific for flanking c onserved target sites. The amplified fragments were cloned or used dir ectly as probes. RNA probes were generated by in vitro transcription o f cloned or amplified rDNA. The probes were labeled by incorporating m odified nucleotides during in vitro DNA amplification or in vitro tran scription or by random priming. The use of in vitro transcribed single -stranded RNA probes instead of double-stranded DNA probes provided st ronger hybridization signals. Group-specific probes were prepared from genomic DNAs or directly from cells of Acinetobacter calcoaceticus, A lcaligenes faecalis, Aeromonas hydrophila, Nannocystis exedens, Pseudo monas aeruginosa, Pseudomonas fluorescens, and Pseudomonas stutzeri.