Description
Wine can potentially be spoiled by the presence of Brettanomyces bruxellensis (the anamorphic stage of Dekkera bruxellensis). This yeast has been identified as the principal source of some volatile compounds such as 4-ethylphenol, 4-ethylguaiacol, and isovaleric acid, which are associated with the typical “Brett” character. Albeit that some winemakers consider low concentrations of these compounds as positive as they contribute to the complexity of the wine, when exceeding greatly the sensory threshold these compounds are generally considered undesired as they contribute to sensory characters associated with “horse stable”, “sweaty saddle”, and “rancidity”.
Because Brettanomyces grows on the skin of grapes, contamination during wine production is unavoidable. Moreover, Brett can be introduced to a winery by fruit flies or contaminated wine barrels. Detection of B. bruxellensis is usually carried out using traditional microbiological techniques based on incubating organisms on selective media. The long incubation time (1-2 weeks) is a real problem, which may lead to serious economic consequences.
This qPCR Detection Kit provides a sensitive and reliable method for the detection of Brettanomyces bruxellensis based on qPCR using specific FAM-labeled probes, requiring only a couple of hours. This immense time reduction allows winemakers to take appropriate action, if needed, much sooner. This kit is compatible with instruments equipped with FAM and ROX channels. The detection limit is approximately 50fg of Brettanomyces DNA, or as little as 102-103 cells per 50ml of wine, with a specificity of 100%.
FEATURES
Fast, Easy and Reliable
LOD: 50 fg of target DNA /102-103 cells per 50ml of wine
Compatible with instruments equipped with FAM and ROX channels
Inclusivity test
A total of 3 Brettanomyces/Dekkera bruxellensis strains were tested, using 2 ng of genomic DNA and all found to be positive.
100% Specificity
A total of 34 non-target species (20 Yeasts, 12 Fungi and 2 Bacteria) related with the target and/or commonly found in wine, were tested in triplicate for exclusivity, using 10ng of purified genomic DNA. None of the testes species showed a positive result.
List of specied used for exclusivity test:
Y101 Brettanomyces/Dekkera anomala, Y102 Brettanomyces/Dekkerra naardenensis, Y001 Saccharomyces cerevisae CECT, Y004 Candida tropicalis PYCC 3097T, Y005 Candida lusitaneae PYCC 2705T, Y006 Candida krusei, Y007 Candida parapsilosis PYCC 2545T, Y008 Candida glabrata PYCC 2418T, Y009 Cryptococcus neoformans, Y010 Trichosporon sp, Y011 Pichia membranifaciens, Y012 Schizosaccharomyces pombe, 4 strains of Y003 Zygosaccharomyces bailii, Y103 Zygosaccharomyces sp, Y104 Zygosaccharomyces bisporus, Y105 Zygosaccharomyces lentus, Y106 Zygosaccharomyces rouxii, F001 Aspergillus niger, F002 Aspergillus flavus, 2 strains of F003 Aspergillus fumigates, F004 Fusarium sp, F005 Trichophyton tonsurans, F006 Sporothrix schenckii, F007 Scopulariopsis brevicaulis, F008 Trichophyton erinacei, F009 Trichonphyton verrucosum, F010 Arthroderma benhamiae, F011 Trichophyton mentagrophytes, B029 Lactobacillus paracasei subsp paracasei CECT 4022T, B030 Oenococcus oeni.
All strains were confirmed by analysis of rRNA genes from purified DNA amplified using universal primers.
Trueness
Trueness of the method was evaluated using 5 positive (artificilally enriched with Brettanomyces/Dekkera bruxellensis) and 2 negative wine samples. All samples were tested in triplicate. All samples presented the epected results for all the replicates. Hence a 0% false positive rate and a 0% false negative rate, corresponding to a trueness of 100%
