Multiple region high resolution melting-based method for accurate differentiation of food-derived yeasts at species level resolution

Yeasts comprise a divergent group of microorganisms playing an important role in foods production. To monitor the production processes, validate authenticity and safety of foods, reliable methods of yeast identification and differentiation are needed. Nowadays, traditional PCR/sequencing-based methods are replaced by rapid tech niques not requiring post-PCR processing. In the present study, we developed a three region-based qPCR-HRM protocol, for rapid differentiation of yeast species isolated from food products. The three targeted fragments (26S rDNA, 18S rDNA, ITS) were carefully analyzed to dock the primers at inter-species conservative regions, flanking polymorphic regions of ~200 bp. Thirty eight yeast strains were used as a training material. The collection of yeast spanned Pichia, Clavispora, Candida, Yarrowia, Kluyveromyces, Saccharomyces, and Wickerhamomyces genera. MALDI-TOF mass spectrometry and conventional rDNA sequencing were used for validation. Conducted studies demonstrated that when used individually, each of the three regions analyzed by qPCR-HRM possessed its own yeast species-specific limitations, sometimes leading to inaccurate taxonomic classification. On the other hand, simultaneous analysis of the three proposed regions resulted in rapid and adequate differentiation of all the yeast strains at species-level resolution.