Use of propidium monoazide for the enumeration of viable Brettanomyces bruxellensis in wine and beer by quantitative PCR

• PMA-qPCR rapidly enumerates viable cells of Brettanomyces bruxellensis in wine/beer.
• Lowest detection limit reached until now for live B. bruxellensis detection.
• PMA-qPCR avoids time consuming and expensive step of RNA extraction.
• Detection limit in red and white wine is 0.83 and 0.63 log cfu/ml, respectively.
• Detection limit in beer 0.23 log cfu/mL.

Brettanomyces bruxellensis is a current problem in winemaking all over the world, and the question if B. bruxellensis has a positive or negative impact on wine is one of the most controversial discussions in the world. The presence of live B. bruxellensis cells represents the risk of growth and an increase in cell numbers, which is related to the potential production of volatile phenols. In this work, the optimisation of a PMA-quantitative PCR (qPCR) method to enumerate only viable cells was carried out using the standard strain B. bruxellensis DSMZ 70726. The obtained detection limits were 0.83 log CFU/mL in red wine, 0.63 log CFU/mL in white wine and 0.23 log CFU/mL in beer.Moreover, the quantification was also performed by Reverse Transcription quantitative PCR (RT-qPCR), and the results showed a higher detection limit for all of the trials.