0000000000400591
AUTHOR
Verónica Soares-santos
Improved detection and enumeration of yeasts in wine by Cells-qPCR
Abstract Quantitative PCR by directly sampling (Cells-qPCR) has been adapted to detect and quantify total yeasts, and B. bruxellensis, S. cerevisiae and Z. bailii species, in grape musts and wines. To increase assay sensitivity, the effects of a previous cell wall lysis, by both enzymatic and mechanical methods, were evaluated. Cell wall disruption by mechanical methods showed the best results to enhance assay sensitivity. Numerous standard curves were constructed by mechanically lysed cells in culture medium, and in white and red grape musts and wines. Good regression values (>0.99) and efficiencies (>0.99) were obtained, and it was possible to detect one single cell per reaction in all th…
Direct and Rapid Detection and Quantification of Oenococcus oeni Cells in Wine by Cells-LAMP and Cells-qLAMP
Fast detection and enumeration of Oenococcus oeni in winemaking are necessary to determine whether malolactic fermentation (MLF) is likely to be performed or not and to decide if the use of a commercial starter is needed. In other wines, however, performing MLF can be detrimental for wine and should be avoided. The traditional identification and quantification of this bacteria using culture-dependent techniques in wine-related matrices require up to 14 days to yield results, which can be a very long time to perform possible enological operations. Loop-mediated isothermal amplification (LAMP) is a novel culture-independent technique that amplifies nucleic acid sequences under isothermal cond…
Cells-qPCR as a direct quantitative PCR method to avoid microbial DNA extractions in grape musts and wines.
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 observe…