Real-Time PCR Assay for Detection and Enumeration of<i>Dekkera bruxellensis</i>in Wine

Phister, Trevor G.; Mills, David A.

doi:10.1128/aem.69.12.7430-7434.2003

Cited by 106 publications

(83 citation statements)

References 22 publications

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“…Absolute quantification methods by QPCR often use plasmid dilutions (Fontaine and Guillot 2002;Galluzzi et al 2004) or laboratory cultures (Bowers et al 2000;Phister and Mills 2003;Popels et al 2003;Saito et al 2002) to generate a standard curve. Although these standards are relatively easy to prepare, they may not always be appropriate for comparison to natural populations of target cells.…”

Section: Discussionmentioning

confidence: 99%

“…The fractional cycle number (Ct) is calculated for each reaction at a point where the fluorescence signal crosses a certain threshold. Several studies have demonstrated the potential of the methodology for the quantitative analysis of microorganisms (Cullen et al 2002;Fontaine and Guillot 2002;Phister and Mills 2003;Skovhus 2004;Suzuki et al 2000;Vaitomaa et al 2003) including harmful algal bloom (HAB) species (Bowers et al 2000;Galluzzi et al 2004;Gray et al 2003;Popels et al 2003;Saito et al 2002) in environmental samples. The basic approach used in these studies is to generate a standard curve using plasmids with the target DNA sequence or DNA extracted from cultures with known concentrations of the target species.…”

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confidence: 99%

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Improved quantitative real‐time PCR assays for enumeration of harmful algal species in field samples using an exogenous DNA reference standard

Coyne

Handy

Demir

et al. 2005

Limnology & Ocean Methods

137

152

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Quantitative real-time PCR (QPCR) is a powerful and sensitive method for quantitative detection of microorganisms. Application of this methodology for enumeration of harmful algal bloom (HAB) species has the potential to revolutionize our approach to HAB research, making it possible to identify correlations between cell abundances and factors that regulate bloom dynamics. Its application to ecological studies, however, has produced mixed results. QPCR assays typically rely on the generation of standard curves from plasmids or laboratory cultures that may be unrealistic when compared to amplification of DNA extracted from field samples. In addition, existing methods often fail to incorporate controls to assess variability in extraction and amplification efficiencies, or include controls that are sequence-specific and preclude the investigation of multiple species. Here, we describe the development and rigorous analysis of QPCR assays for two HAB species, Chattonella subsalsa and Heterosigma akashiwo, in which we introduce a known concentration of exogenous DNA plasmid into the extraction buffer as a reference standard. Since the target DNA is extracted in the presence of the reference standard, inherent variability in extraction and amplification efficiencies affect both target and standard equally. Furthermore, the reference standard is applicable to QPCR analysis of any microbial species. Using environmental bloom samples as calibrators, we evaluated the accuracy of the comparative Ct method for enumeration of target species in several field samples. Our investigation demonstrates that the comparative Ct method with an exogenous DNA reference standard provides both accurate and reproducible quantification of HAB species in environmental samples.

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Improved quantitative real‐time PCR assays for enumeration of harmful algal species in field samples using an exogenous DNA reference standard

Coyne

Handy

Demir

et al. 2005

Limnology & Ocean Methods

137

152

View full text Add to dashboard Cite

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“…Strain A was a reference strain isolated from a distillery plant 37 and identified by the Institute of Hygiene and Epidemiology of Brussels (THEM) as B. bruxellensis strain 6037. A PCR assay, using primers designed to the 26S rRNA gene was used to confirm that the isolated strains belonged to B. bruxellensis species 29 . Specific primers were DBRUXF: 5'-GGATGGGTGCACCTGGTT-TACAC-3' and DBRUXR: 5'-GAAGGGCCACATTCA-CGAACCCCG -3'.…”

Section: Microorganismsmentioning

confidence: 99%

Diversity in Spoilage Yeast Dekkera/Brettanomyces bruxellensis Isolated from French Red Wine. Assessment During Fermentation of Synthetic Wine Medium

Barbin

Cheval²,

Gilis³

et al. 2008

Journal of the Institute of Brewing

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J. Inst. Brew. 114(1), 69-75, 2008Yeast Brettanomyces bruxellensis is a contaminant found worldwide and is responsible for red wine spoilage due to the development of animal and phenolic off-odours. During this study, 24 Brettanomyces bruxellenis isolates were obtained from red wine samples from two French wineries and these were discriminated as 23 strains. Nine strains coming from 2 wineries and 4 vintages were cultivated in synthetic wine medium for 1500 hours and they gave nine different behaviours. Four main growth patterns (with different growth steps and durations) and three main different sugar consumption profiles were obtained. Glucose and fructose were not limiting substrates for all strains. The production level of 4-ethylphenol was found to vary from strain to strain (from 0.350 to 2.773 mg L -1 ) and was independent of the biomass concentration. Some strains presented a coupled-togrowth production of volatile phenols, others did not. This study showed that different strains of Brettanomyces bruxellensis behaved differently, one from another, under the given conditions taking into consideration several aspects. The results thus demonstrate a large intraspecific diversity.

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“…Due to the advantages afforded by the technique, it was later extended to yeasts in the clinical setting (1,22,27,29) and in food technology (2,3). Currently, the detection of wine spoil-age yeast using this method is limited to the species Dekkera bruxellensis (21). In the present work, we have developed a real-time PCR to detect and enumerate S. cerevisiae directly in wine samples using the nonspecific fluorescent dye SYBR green.…”

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confidence: 99%

Rapid Identification and Enumeration of Saccharomyces cerevisiae Cells in Wine by Real-Time PCR

Martorell

Querol

Fernández-Espinar

2005

Appl Environ Microbiol

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Despite the beneficial role of Saccharomyces cerevisiae in the food industry for food and beverage production, it is able to cause spoilage in wines. We have developed a real-time PCR method to directly detect and quantify this yeast species in wine samples to provide winemakers with a rapid and sensitive method to detect and prevent wine spoilage. Specific primers were designed for S. cerevisiae using the sequence information obtained from a cloned random amplified polymorphic DNA band that differentiated S. cerevisiae from its sibling species Saccharomyces bayanus, Saccharomyces pastorianus, and Saccharomyces paradoxus. The specificity of the primers was demonstrated for typical wine spoilage yeast species. The method was useful for estimating the level of S. cerevisiae directly in sweet wines and red wines without preenrichment when yeast is present in concentrations as low as 3.8 and 5 CFU per ml. This detection limit is in the same order as that obtained from glucose-peptone-yeast growth medium (GPY). Moreover, it was possible to quantify S. cerevisiae in artificially contaminated samples accurately. Limits for accurate quantification in wine were established, from 3.8 ؋ 10 5 to 3.8 CFU/ml in sweet wine and from 5 ؋ 10 6 to 50 CFU/ml in red wine.Wine can become a growth substrate for a range of undesirable yeast species, both during and after fermentation. Uncontrolled yeast growth at either of these two stages can alter the chemical composition of wine, detracting from its sensory properties of appearance, aroma, and flavor. If these faults are severe, the wine is rejected by consumers. Thus, wine spoilage constitutes an important concern to wine producers.It is well known that Saccharomyces cerevisiae plays a beneficial role in wine fermentation, in which it is the predominant species. Nevertheless, S. cerevisiae is able to spoil wine after fermentation if the yeast is not properly eliminated or if the bottle is contaminated by yeast cells present in the wine bottling line or in the cork. S. cerevisiae is an important spoilage yeast because it resists high ethanol concentrations. It has been found mainly in sweet wines, where fermentable sugar can support growth, and also in semidry bottled wines.It is important to detect spoilage yeasts quickly to enable wineries to intervene rapidly and effectively. Methods based on PCR appear to be the best alternative. In the case of S. cerevisiae, several methods have been shown suitable for the detection of this species. This is the case of the restriction analysis of the rRNA region spanning the 5.8S gene and the two internal transcribed spacers (ITSs) (5.8S-ITS region). The amplification patterns of S. cerevisiae when its DNA is digested with the endonuclease CfoI, HaeIII, or HinfI identify this species accurately. These data are summarized in references 10 and 13 and are available online (http://www.yeast-id.com). Masneuf et al. (18) developed a similar system based on PCR amplification and subsequent restriction analysis using the nuclear gene MET2. These author...

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Real-Time PCR Assay for Detection and Enumeration ofDekkera bruxellensisin Wine

Cited by 106 publications

References 22 publications

Improved quantitative real‐time PCR assays for enumeration of harmful algal species in field samples using an exogenous DNA reference standard

Improved quantitative real‐time PCR assays for enumeration of harmful algal species in field samples using an exogenous DNA reference standard

Diversity in Spoilage Yeast Dekkera/Brettanomyces bruxellensis Isolated from French Red Wine. Assessment During Fermentation of Synthetic Wine Medium

Rapid Identification and Enumeration of Saccharomyces cerevisiae Cells in Wine by Real-Time PCR

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