PCR-based preparation of 23S rRNA-targeted group-specific polynucleotide probes.

AUTOR(ES)

Ludwig, W

RESUMO

DNA coding for a variable region within domain III of bacterial 23S rRNA was used as the target for group-specific polynucleotide hybridization probes. The corresponding rDNA was amplified in vitro by the PCR technique in combination with a pair of primers specific for flanking conserved target sites. The amplified fragments were cloned or used directly as probes. RNA probes were generated by in vitro transcription of cloned or amplified rDNA. The probes were labeled by incorporating modified nucleotides during in vitro DNA amplification or in vitro transcription or by random priming. The use of in vitro transcribed single-stranded RNA probes instead of double-stranded DNA probes provided stronger hybridization signals. Group-specific probes were prepared from genomic DNAs or directly from cells of Acinetobacter calcoaceticus, Alcaligenes faecalis, Aeromonas hydrophila, Nannocystis exedens, Pseudomonas aeruginosa, Pseudomonas fluorescens, and Pseudomonas stutzeri.

Documentos Relacionados

• Identification of Whole Fixed Bacterial Cells with Nonradioactive 23S rRNA-Targeted Polynucleotide Probes
• Identification of lactococci and enterococci by colony hybridization with 23S rRNA-targeted oligonucleotide probes.
• Variations of Bacterial Populations in Human Feces Measured by Fluorescent In Situ Hybridization with Group-Specific 16S rRNA-Targeted Oligonucleotide Probes
• Flow cytometric analysis of activated sludge with rRNA-targeted probes.
• Identifying members of the domain Archaea with rRNA-targeted oligonucleotide probes.