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 fo r the type strains of the validly described Enterococcus species, Melissoco ccus pluton and Tetragenococcus halophilus. A comprehensive set of rRNA tar geted specific oligonucleotide hybridization probes was designed according to the multiple probe concept. In silico probe design and evaluation was pe rformed using the respective tools of the ARB program package in combinatio n with the ARE databases comprising the currently available 16S as well as 23S rRNA primary structures. The probes were optimized with respect to thei r application for reverse hybridization in microplate format. The target co mprising 16S and 23S rDNA was amplified and labeled by PCR (polymerase chai n reaction) using general primers targeting a wide spectrum of bacteria. Al ternatively, amplification of two adjacent rDNA fragments of enterococci wa s 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 c ontent. The optimized probe set was used to analyze enriched drinking water samples as well as original samples from waste water treatment plants.