Cells-qPCR as a direct quantitative PCR method to avoid microbial DNA extractions in grape musts and wines

Abstract

A novel quantitative PCR assay called Cells-qPCR has been developed for the rapid detection and quantification of yeasts, lactic acid bacteria (LAB) and acetic acid bacteria (AAB) directly from grape must and wine that does not require DNA extraction. The assay was tested on Brettanomyces bruxellensis, Saccharomyces cerevisiae, Lactobacillus plantarum, Oenococcus oeni, Acetobacter aceti and Gluconobacter oxydans in culture media, and in white and red grape musts and wines. Standard curves were constructed from DNA and cells for the six target species in all the matrices. Good efficiencies were obtained for both when comparing DNA and cells standard curves. No reaction inhibition was observed between matrices for each species. Cells quantification was linear over a range of cell concentrations (7, 5 or 4 orders of magnitude) and detected as few as one cell per reaction in all the matrices. The developed Cells-qPCR assay is a robust, reliable, fast and specific method to detect and quantify different yeasts, LAB and AAB species in grape must and wine that avoids DNA extraction and overcomes the presence of inhibitors like polyphenols and ethanol.