4.3.2. Molecular Identification of Yeasts

Purified yeast cultures were lyophilized and weighed. Genomic DNA extraction was performed using the Plant and Fungi DNA purification Kit (EURx, Gdansk, Poland), according to the manufacturer’s recommendation. The concentration of genomic DNA for each sample was measured using NanoDrop 1000 UV–Visible Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA), and the DNA extracts were stored at −20 °C. Polymerase chain reactions (PCRs) were done in 50 µL aliquots using C-1000 thermal cyclers (Bio-Rad, Hercules, CA, USA). Each PCR was conducted using 1.25 U of Color Taq DNA polymerase (EURx, Gdansk, Poland), 5 µL of 10× Pol Buffer B, 0.2 mM of each dNTP, 0.5 µM of forward/reverse primers, and about 20 ng of yeast DNA. PCR conditions were followed: 5 min at 95 °C, 35 cycles of 1 min at 95 °C, 1 min at 56 °C (annealing), and 1 min at 72 °C, and the final extension 7 min at 72 °C. For the PCR amplification of a D1/D2 region of 26S rRNA gene, the primers NL1 (5′-GCATATCAATAAGCGGAGGAAAAG-3′) and NL4 (5′-GGTCCGTGTTTCAAGACGG-3′) [65] were used. Both yeasts [66] and filamentous fungi [67,68] were identified based on amplification and sequencing of the large subunit ribosomal RNA sequences (D1/D2 domains). Amplicons were separated in 1.5% agarose gel (EURx, Gdansk, Poland) with a SimplySafe nucleic acid stain (EURx, Gdansk, Poland).

For sequence analysis, obtained amplicons were purified as described earlier by Kozłowska et al. [65]. Afterward, the purified amplicons were labeled using a reverse primer (NL4) and the BigDye Terminator 3.1 kit (Applied Biosystems, Foster City, CA, USA), according to Tomczyk et al. [69]. Sequences were analyzed using the BLASTn algorithm and compared with reference sequences from the GenBank database.