A nested array of rRNA targeted probes for the detection and identification of enterococci by reverse hybridization.

Complete 23S and almost complete 16S rRNA gene sequences were determined for the type strains of the validly described Enterococcus species, Melissococcus pluton and Tetragenococcus halophilus. A comprehensive set of rRNA targeted specific oligonucleotide hybridization probes was designed according to the multiple probe concept. In silico probe design and evaluation was performed using the respective tools of the ARB program package in combination with the ARB databases comprising the currently available 16S as well as 23S rRNA primary structures. The probes were optimized with respect to their application for reverse hybridization in microplate format. The target comprising 16S and 23S rDNA was amplified and labeled by PCR (polymerase chain reaction) using general primers targeting a wide spectrum of bacteria. Alternatively, amplification of two adjacent rDNA fragments of enterococci was performed by using specific primers. In vitro evaluation of the probe set was done including all Enterococcus type strains, and a selection of other representatives of the gram-positive bacteria with a low genomic DNA G+C content. The optimized probe set was used to analyze enriched drinking water samples as well as original samples from waste water treatment plants.

Application of 23S rDNA-targeted oligonucleotide probes specific for enterococci to water hygiene control.

Identification of enterococci species by hybridization with recently designed species-specific and group-specific 23S rDNA-targeted oligonucleotide probes was superior to results obtained with a common biochemical test panel. Considering these findings, a molecular biological procedure for the detection of enterococci in water samples was developed. A short enrichment is followed by an amplification step and a hybridization reaction in microtiter plate format. The detection limit is about 1 CFU/ml, and results are available within 26 h.