Design and Performance Testing of a DNA Extraction Assay for Sensitive and Reliable Quantification of Acetic Acid Bacteria Directly in Red Wine Using Real Time PCR.

Cédric Longin,Michèle Guilloux-Benatier,Hervé Alexandre

doi:10.3389/fmicb.2016.00831

Cédric Longin, Michèle Guilloux-Benatier + Show 1 more

Open Access

https://doi.org/10.3389/fmicb.2016.00831

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Abstract

Although strategies exist to prevent AAB contamination, the increased interest for wines with low sulfite addition leads to greater AAB spoilage. Hence, there is a real need for a rapid, specific, sensitive, and reliable method for detecting these spoilage bacteria. All these requirements are met by real time Polymerase Chain Reaction (or quantitative PCR; qPCR). Here, we compare existing methods of isolating DNA and their adaptation to a red wine matrix. Two different protocols for isolating DNA and three PCR mix compositions were tested to select the best method. The addition of insoluble polyvinylpolypyrrolidone (PVPP) at 1% (v/v) during DNA extraction using a protocol succeeded in eliminating PCR inhibitors from red wine. We developed a bacterial internal control which was efficient in avoiding false negative results due to decreases in the efficiency of DNA isolation and/or amplification. The specificity, linearity, repeatability, and reproducibility of the method were evaluated. A standard curve was established for the enumeration of AAB inoculated into red wines. The limit of quantification in red wine was 3.7 log AAB/mL and about 2.8 log AAB/mL when the volume of the samples was increased from 1 to 10 mL. Thus, the DNA extraction method developed in this paper allows sensitive and reliable AAB quantification without underestimation thanks to the presence of an internal control. Moreover, monitoring of both the AAB population and the amount of acetic acid in ethanol medium and red wine highlighted that a minimum about 6.0 log cells/mL of AAB is needed to significantly increase the production of acetic acid leading to spoilage.

Highlights

Acetic Acid Bacteria (AAB) species typically associated with grapes and must is Gluconobacter oxydans (G. oxydans) which prefers a sugar rich environment (Joyeux et al, 1984; Bartowsky and Henschke, 2008)
Cell quantification by qPCR in red wine is difficult since various qPCR inhibitors such as polyphenols and polysaccharides are abundant, thereby increasing the risk of false negative results (Demeke and Jenkins, 2009) and making the amplification of genetic material challenging. qPCR has been used to quantify AAB in wine (González et al, 2006; Andorrà et al, 2008; Torija et al, 2010; Valera et al, 2013), the authors of these studies did not take into account extraction efficiency or the presence of inhibitors, nor did they use any control process
In our study, we developed a qPCR method which allows the reliable quantification of AAB in red wine

Summary

Introduction

Acetic Acid Bacteria (AAB) species typically associated with grapes and must is Gluconobacter oxydans (G. oxydans) which prefers a sugar rich environment (Joyeux et al, 1984; Bartowsky and Henschke, 2008). Many studies have reported that plate counting is not appropriate for estimating AAB populations in stressful environments like wine (Sievers et al, 1992; Sokollek et al, 1998; Millet and Lonvaud-Funel, 2000; Bartowsky et al, 2003; Trcek, 2005). Independent culture quantification techniques using Denaturing Gradient Gel Electrophoresis (De Vero et al, 2006), Temperature Gradient Gel Electrophoresis (Ilabaca et al, 2008), or epifluorescence have been reported (Mesa et al, 2003; BaenaRuano et al, 2006) These latter authors quantified AAB in vinegar fermentation using viability (i.e., measurement of cell membrane permeability) and vitality (i.e., measurement of cell enzymatic activity) dyes. Different DNA extraction protocols were compared in order to remove wine inhibitors and assess the sensitivity, specificity, and reproducibility of the method

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