Fast protocols for the 5S rDNA and ITS-2 based identification of Oenococcus oeni.

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.