Development and application of a real-time PCR-based assay to enumerate total yeasts and Pichia anomala, Pichia guillermondii and Pichia kluyveri in fermented table olives

The objective of this work was to develop a real-time quantitative PCR (qPCR) assay to directly detect and quantify the total number of yeasts besides three species (Pichia anomala, Pichia guillermondii and Pichia kluyveri) associated to table olives. For this purpose, a real-time PCR protocol targeted to the region of the internal transcribed spacers ITS (rDNA) and the conserved sequences of the variable D1/D2 domains of the 26S rRNA gene, respectively, was developed. This assay allowed to unequivocally distinguish the three species from other yeasts and lactic acid bacteria generally associated to table olives fermentation. qPCR performed well both with purified DNA and DNA extracted from olive brines. A reverse transcription-qPCR (RT-qPCR) assay from rRNA aimed to viable yeast quantification was also performed. To evaluate the effectiveness of the technique, the qPCR results were compared with those obtained by a plate count approach in synthetic medium. The small standard errors provided this assay reproducible and robust. qPCR efficiently enumerated cells at concentrations of as low as 102 CFU ml−1 when standard curve was derived both from cells growing in a syntetic medium and in brines. The quantification method for total yeasts and P. anomala, P. guillermondii, P. kluyveri in table olive brines applied in this work was specific, reproducible, sensitive and fast.