rRNA probe-based cell fishing of bacteria

We have developed a new, cultivation-independent, fast and flexible method for the rRNA-targeted probe-based enrichment of bacteria. The target cells were labelled by in situ hybridization with biotinylated polyribonucleotide probes. These probes were generated by in vitro transcription of amplified rDNA of a variable region in domain III of the 23S rRNA molecules. The pro bes were about 300 nucleotides in length and were labelled by incorporation of biotin-UTP during the transcription. Probes were hybridized with bacter ial cells and incubated with paramagnetic streptavidin-coated particles. Th e labelled target cells can be separated in a column filled with steel wool inserted into the field of a permanent magnet. Unlabelled, non-target cell s pass through the column, whereas labelled cells are retained. They were e luted from the column after removal of the magnetic field. Up to now, the m ethod has been tested with mixtures of different pure cultures. For the fir st time, transcript probes have been used for the labelling of the target c ells and for their specific separation, The enrichment of the target cells can be monitored by a streptavidin-fluorescein staining of the biotinylated target cells and/or by a subsequent in situ hybridization with fluorescent ly labelled oligonucleotide probes. Enrichment rates of up to 90-fold, depe nding on the original abundance of the cells of interest, could be determin ed. To demonstrate that the sorted cells were amenable to molecular analysi s, we amplified and sequenced a part of the tuf gene of enriched Acinetobac ter calcoaceticus cells.