Rapid sourdough yeast identification using panfungal PCR combined with high resolution melting analysis

The microbial composition of the sourdough starter affects the sourdough bread properties. Therefore, it is crucial to find a tool for rapid, time-saving, and economical identification of the sourdough microbiota. We focused on the rapid identification of sourdough yeasts. We designed a panfungal real time-PCR targeting the ITS2 region (ITS-amplicon) and a fragment of D1/D2 region of 26S rRNA gene (U-amplicon) and used high resolution melting analysis (HRM) for subsequent species identification. The sensitivity and specificity of our method were tested on the reference yeast cultures. We obtained divergent melting peaks (Tm). The further analysis of melt curves suggests the possibility to discriminate yeasts on the genus- and some on species-specific level in the mixed sample. The applicability of this method in routine practice was evaluated on nine sourdough samples. Revealed melt curves of U-amplicons were predominantly characteristic of the sourdough. The evaluation of the Tm and the shape of the melt curve was used to assess the sourdough yeasts. Additionally, using the HRM-PCR method the contamination with the ergot fungus DNA was revealed. Our data showed HRM-PCR is a simple, rapid, and inexpensive tool useful in identifying sourdough yeasts.