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

Soares-Santos, Verónica; Pardo, Isabel; Ferrer, Sergi

doi:10.1016/j.ijfoodmicro.2017.08.019

Cited by 11 publications

(7 citation statements)

References 29 publications

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“…Tubes were centrifuged, DNA was extracted as previously described and used to construct the calibration curve. In this way, the presence of contaminants, such as tannins and other polyphenols, on the efficiency of the qPCR reaction was taken into account [31]. Moreover, it was possible to verify the presence of any variations in the extraction efficiency due to the different concentrations of the cells, evaluating the linearity of the observations.…”

Section: Real Time Pcr Quantification Of Starmerella Bacillarismentioning

confidence: 99%

Starmerella bacillaris Released in Vineyards at Different Concentrations Influences Wine Glycerol Content Depending on the Vinification Protocols

Nadai

Duarte

Sica

et al. 2022

Foods

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Starmerella bacillaris is a non-Saccharomyces yeast proposed for must fermentation together with Saccharomyces cerevisiae because of its high glycerol and moderate volatile acidity production. Furthermore, it was demonstrated that the same S. bacillaris strains that possess interesting technological properties exhibited antifungal activity against Botrytis cinerea, suggesting the release of this yeast in the vineyard. To obtain a positive effect during the following winemaking process, the maintenance of suitable concentrations of S. bacillaris is essential. Therefore, to obtain information on the survival of S. bacillaris, a small-scale field trial was performed. One week before the harvest, two different concentrations of S. bacillaris (106 and 107 cells/mL) were sprayed on pinot grigio bunches, and the strain concentration was monitored by means of qPCR during the subsequent fermentation process. In addition, the combined effect of different winemaking techniques was evaluated, i.e., the vinification of juice, juice with marc and cryomaceration treatment. Results demonstrated that, under the tested conditions, S. bacillaris released in the vineyard remained viable for one week on grape bunches and increased glycerol content during the subsequent fermentation process. Different vinification protocols influenced cell concentrations. In particular, the cryomaceration treatment, due to the use of low temperature, supported S. bacillaris growth due to its cryotolerant aptitude. The collected data open new perspectives on the control of alcoholic fermentation, involving both vineyard and cellar management.

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Section: Real Time Pcr Quantification Of Starmerella Bacillarismentioning

confidence: 99%

Starmerella bacillaris Released in Vineyards at Different Concentrations Influences Wine Glycerol Content Depending on the Vinification Protocols

Nadai

Duarte

Sica

et al. 2022

Foods

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“…The similar results were also reported in other studies. For example, the standard curve of B. bruxellensis , O. oeni and A. acet using qPCR method exhibited a greater cell concentration range than CELL-qPCR assay ( Soares-Santos et al, 2017 ). In the present study, all the amplification methods showed the good amplification efficiencies (0.8 to 1.2) with qPCR slightly outperforming the other two assays in the cells ( Table 2 ).…”

Section: Resultsmentioning

confidence: 99%

“…This technique eliminates the DNA extraction process and directly utilizes cells as template for the qPCR amplification. This microbial population counting method shortens the reaction time and lowers the reaction cost ( Soares-Santos et al, 2017 , 2018 ). To our best knowledge, CELL-qPCR method, coupled with the PMA treatment, has not been studied on microbial kinetics in wine-making process.…”

Section: Introductionmentioning

confidence: 99%

A rapid and reliable method for the determination of Lactiplantibacillus plantarum during wine fermentation based on PMA-CELL-qPCR

et al. 2023

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Real-time monitoring of microbial dynamics during fermentation is essential for wine quality control. This study developed a method that combines the fluorescent dye propidium monoazide (PMA) with CELL-qPCR, which can distinguish between dead and live microbes for Lactiplantibacillus plantarum. This method could detect the quantity of microbes efficiently and rapidly without DNA extraction during wine fermentation. The results showed that (1) the PMA-CELL-qPCR enumeration method developed for L. plantarum was optimized for PMA treatment concentration, PMA detection sensitivity and multiple conditions of sample pretreatment in wine environment, and the optimized method can accurately quantify 104–108 CFU/mL of the target strain (L. plantarum) in multiple matrices; (2) when the concentration of dead bacteria in the system is 104 times higher than the concentration of live bacteria, there is an error of 0.5–1 lg CFU/mL in the detection results. The optimized sample pretreatment method in wine can effectively reduce the inhibitory components in the qPCR reaction system; (3) the optimized PMA-CELL-qPCR method was used to monitor the dynamic changes of L. plantarum during the fermentation of Cabernet Sauvignon wine, and the results were consistent with the plate counting method. In conclusion, the live bacteria quantification method developed in this study for PMA-CELL-qPCR in L. plantarum wines is accurate in quantification and simple in operation, and can be used as a means to accurately monitor microbial dynamics in wine and other fruit wines.

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“…It uses two external and two internal primers, allowing the direct detection B. bruxellensis in wines without strain isolation [ 9 ]. More recently, a quantitative PCR by direct sampling (Cells-qPCR) has been used to detect and quantify yeasts in grape, must, and wine samples [ 24 ]. Cells-qPCR thus results a fast and sensitive technique.…”

Section: Discussionmentioning

confidence: 99%

Rapid Detection of Brettanomyces bruxellensis in Wine by Polychromatic Flow Cytometry

Bellis

Stefano

Simeone

et al. 2022

IJMS

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Brettanomyces bruxellensis is found in several fermented matrices and produces relevant alterations to the wine quality. The methods usually used to identify B. bruxellensis contamination are based on conventional microbiological techniques that require long procedures (15 days), causing the yeast to spread in the meantime. Recently, a flow cytometry kit for the rapid detection (1–2 h) of B. bruxellensis in wine has been developed. The feasibility of the method was assessed in a synthetic medium as well as in wine samples by detecting B. bruxellensis in the presence of other yeast species (Saccharomyces cerevisiae and Pichia spp.) and at the concentrations that produce natural contaminations (up to 105 cells/mL), as well as at lower concentrations (103–102 cells/mL). Wine samples naturally contaminated by B. bruxellensis or inoculated with four different strains of B. bruxellensis species together with Saccharomyces cerevisiae and Pichia spp., were analyzed by flow cytometry. Plate counts were carried out in parallel to flow cytometry. We provide evidence that flow cytometry allows the rapid detection of B. bruxellensis in simple and complex mixtures. Therefore, this technique has great potential for the detection of B. bruxellensis and could allow preventive actions to reduce wine spoilage.

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

Cited by 11 publications

References 29 publications

Starmerella bacillaris Released in Vineyards at Different Concentrations Influences Wine Glycerol Content Depending on the Vinification Protocols

Starmerella bacillaris Released in Vineyards at Different Concentrations Influences Wine Glycerol Content Depending on the Vinification Protocols

A rapid and reliable method for the determination of Lactiplantibacillus plantarum during wine fermentation based on PMA-CELL-qPCR

Rapid Detection of Brettanomyces bruxellensis in Wine by Polychromatic Flow Cytometry

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