title: Species-specific identification of Dekkera/Brettanomyces yeasts by fluorescently labeled DNA probes targeting the 26S rRNA.
abstract: Sequencing of the complete 26S rRNA genes of all Dekkera/Brettanomyces species colonizing different beverages revealed the potential for a specific primer and probe design to support diagnostic PCR approaches and FISH. By analysis of the complete 26S rRNA genes of all five currently known Dekkera/Brettanomyces species (Dekkera bruxellensis, D. anomala, Brettanomyces custersianus, B. nanus and B. naardenensis), several regions with high nucleotide sequence variability yet distinct from the D1/D2 domains were identified. FISH species-specific probes targeting the 26S rRNA gene's most variable regions were designed. Accessibility of probe targets for hybridization was facilitated by the construction of partially complementary 'side'-labeled probes, based on secondary structure models of the rRNA sequences. The specificity and routine applicability of the FISH-based method for yeast identification were tested by analyzing different wine isolates. Investigation of the prevalence of Dekkera/Brettanomyces yeasts in the German viticultural regions Wonnegau, Nierstein and Bingen (Rhinehesse, Rhineland-Palatinate) resulted in the isolation of 37 D. bruxellensis strains from 291 wine samples.
authors.affiliation: Institute of Microbiology and Wine Research, Johannes Gutenberg University, Mainz, Germany. roederc@uni-mainz.de
authors.forename: Christoph
authors.initials: C
authors.lastname: Röder
authors.forename: Helmut
authors.initials: H
authors.lastname: König
authors.forename: Jürgen
authors.initials: J
authors.lastname: Fröhlich
mesh_terms: D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D004271:DNA, Fungal / Q000235:genetics; D017404:In Situ Hybridization, Fluorescence; D008969:Molecular Sequence Data; D016533:Mycological Typing Techniques; D009690:Nucleic Acid Conformation; D015341:Nucleic Acid Probes / Q000235:genetics; D010802:Phylogeny; D012335:RNA, Ribosomal / Q000032:analysis* / Q000235:genetics; D004718:Saccharomycetales / Q000145:classification* / Q000235:genetics* / Q000302:isolation & purification; D017422:Sequence Analysis, DNA; D013045:Species Specificity; D014920:Wine / Q000382:microbiology
chemical_list: D017931:DNA Primers; D004271:DNA, Fungal; D015341:Nucleic Acid Probes; D012335:RNA, Ribosomal; C065096:RNA, ribosomal, 26S