Fast protocols for the 5S rDNA and ITS-2 based identification ofOenococcus oeni

Abstract

To identify specific marker sequences for the rapid identification of Oenococcus oeni, we sequenced the 23S-5S internal transcribed spacer (ITS-2) region and the 5S rDNA of five different O. oeni strains and three phylogenetically related lactic acid bacteria (LAB). Comparative analysis revealed 100% identity among the ITS-2 region of the O. oeni strains and remarkable differences in length and sequence compared to related LAB. These results enabled us to develop a primer set for a rapid PCR-identification of O. oeni within three hours. Moreover, the comparison of the 5S rDNA sequences and the highly conserved secondary structure provided the template for the design of three fluorescence-labeled specific oligonucleotides for fluorescence in situ hybridization (FISH). These probes are partial complementary to each other. This feature promotes the accessibility to the target sequence within the ribosome and enhances the fluorescence signal. For the rapid identification of Oenococci both the 5S rRNA gene and the ITS-2 region are useful targets.

Species-specific identification ofDekkera/Brettanomycesyeasts by fluorescently labeled DNA probes targeting the 26S rRNA

“…FISH of whole cells is particularly suitable for the direct detection and identification of microorganisms in their natural environments. Detection and/or identification of wine-related microorganisms by fluorescently labeled rRNA targeting probes has been already performed successfully in previous studies (Fröhlich et al, 2003;Hirschhäuser et al, 2005), including Dekkera/Brettanomyces and other yeasts (Stender et al, 2001;Xufre et al, 2006). This is the first study to provide extensive information about whole LSU rRNA gene sequence variability among all species of the genus Dekkera/Brettanomyces, and suggests a distinct and specific FISH probe design that takes into account the secondary structure of the LSU rRNA.…”

“…FISH of whole cells is particularly suitable for the direct detection and identification of microorganisms in their natural environments. Detection and/or identification of wine‐related microorganisms by fluorescently labeled rRNA targeting probes has been already performed successfully in previous studies (Fröhlich et al , 2003; Hirschhäuser et al , 2005), including Dekkera / Brettanomyces and other yeasts (Stender et al , 2001; Xufre et al , 2006).…”

Detection and identification of microorganisms in wine: a review of molecular techniques

“…Blasco et al [8] designed oligonucleotide probes that target the 16S rDNA subunit to directly detect L. brevis, L. collinoides, L. coryniformis, L. farciminis, L. mali, L. casei/paracasei, L. zeae, O. oeni, P. damnosus, and P. parvulus, from wine samples. Hirschhäuser et al [48] used the 5S rDNA as a target to distinguish O. oeni from closely related lactic acid bacteria species. The 5S rDNA subunit of O. oeni is highly conserved and has a different sequence than other phylogenetically related lactic acid bacteria.…”

“…It was shown, however, that using only one probe with this method resulted in rapid bleaching of the fluorophore, while using multiple probes showed an increase in fluorescent yield. Overall, three probes for the 5S rDNA target were designed and utilized for detection of O. oeni [48].…”

“…These probes target the D1/D2 domain of the large subunit (26S) rDNA. To improve probe hybridization, 'side' probes, which are partially complementary or directly adjacent to the probe, were used to enhance the accessibility of highly structured RNA, a common problem with rDNA-targeted probes [48,97].…”