Analysis of several methods for the extraction of high quality DNA from acetic acid bacteria in wine and vinegar for characterization by PCR-based methods
Introduction
Acetic acid bacteria (AAB) are important microorganisms in the food industry because of their ability to oxidize sugars and alcohols. While their activity sometimes causes food spoilage (in wines, beer, grapes and other fruits, etc.), they are also the main actors in the production of vinegar. Correct and fast enumeration and identification protocols are needed to suitably control the process. One of the biggest hurdles when studying AAB is their cultivation, growth and maintenance in pure culture, especially when isolated from high ethanol and/or acetic acid sources such as wine and vinegar (Entani et al., 1985, Sievers et al., 1992, Gullo et al., 2006). These extreme media are responsible for the limited culture recovery of a significant number of microbiota (Sievers et al., 1992, Sokollek et al., 1998, Millet and Lonvaud-Funel, 2000, Trcek, 2005). AAB are considered “fastidious microorganisms”, mainly because of their inability to recover a part of the population that, though viable, in adverse conditions enters into a non-culturable state (McDougald et al., 1998, Fleet, 1999, Millet and Lonvaud-Funel, 2000, Giraffa and Neviani, 2001). With AAB, it has been reported that conventional plate counts are considerably lower than the fluorescence optical counts of microbial cells from industrial acetators (Mesa et al., 2003, Baena-Ruano et al., 2006).
To overcome the disadvantages of culture-dependent methods, therefore, in recent years several molecular techniques based on PCR have been successfully used to detect bacteria without cultivation in foods (reviewed by Fleet, 1999, Giraffa, 2004). However, the validity and robustness of the results obtained from such molecular techniques depend on the efficient recovery of bacterial DNA.
DNA extraction is usually affected by factors such as incomplete cell lysis, DNA sorption to a particular material, coextraction of enzymatic inhibitors and degradation or damage of DNA (Miller et al., 1999). Clearly, the application of a suitable DNA extraction protocol for a specific sample is essential for correct microbial diversity estimation. The DNA extraction method must be simple, quick and efficient. Safety, cost and DNA quality must also be considered. DNA quality is critical because the efficiency of PCR amplification can be reduced by inhibitors from the matrix. DNA extraction has therefore been highlighted as a limitation of culture-independent methods (Abriouel et al., 2006, Cankar et al., 2006).
With wine and vinegar, which are complex matrices, the presence of polysaccharides and a range of polyphenolics (including tannins) can interfere with enzymatic reactions and even degrade the DNA during direct DNA extraction (Do and Adams, 1991, Demeke and Adams, 1992, Wilson, 1997). No standardized DNA extraction method is yet available for wine and vinegar and all described methods involve chemical and enzymatic lysis. These methods often combine detergents, physical disruption, solvent extraction and enzymatic lysis to obtain crude extracts of nucleic acid (Sokollek et al., 1998, Nanda et al., 2001, González et al., 2004, Gullo et al., 2006, Haruta et al., 2006, Ndoye et al., 2006).
To optimize the detection of AAB from wine and vinegar samples and minimize these limitations, we evaluated five nucleic acid extraction methods on the basis of DNA PCR amplification quality and recovery. Amplification of 16S rRNA gene was assessed by conventional PCR and taken as an indicator of good DNA extraction. Real-time quantitative PCR was used to evaluate the reaction efficiency by quantifying DNA recovery.
Section snippets
Bacterial strain and media
The reference strains used in this study were Acetobacter pasteurianus (LMG 1262T) and Gluconacetobacter hansenii (DSMZ 5602T). These strains were grown in 30 ml of Glucose medium (GY: 1% yeast extract; 1% glucose, w/v) (Cultimed, Barcelona, Spain) for 3–5 days at 30 °C. Each day the number of cells per ml was determined by microscopic counting until the population was 108 cell ml− 1, which was the cell size used for DNA extraction. 1 ml volume of the growth medium was centrifuged and the pellet
PCR inhibition assay
To estimate the quality of the DNA obtained from both species before and after treatments with the extraction protocols, we used a PCR system based on amplification of 16S rRNA gene and visualization onto agarose gel (Table 2). To validate the methods, we tested four matrices (white and red wines, commercial and traditional vinegars) using GY medium as control. Our results were similar for both species. Amplification was generally good when DNA extracted from cells grown in GY medium was used.
Discussion
Ideally, DNA extraction methods should be simple, quick and efficient. Choosing an extraction method often involves a trade-off between cost (materials and labor), the optimal yield of DNA and the removal of substances that could influence the PCR reaction (Cankar et al., 2006). It is necessary, therefore, to carefully validate the suitability of a method for specific samples. The main criteria for this validation are based on quantitative and qualitative analysis.
Today the direct lysis method
Acknowledgments
We are grateful for financial assistance from the Spanish Government (Project AGL 2004-07494-C02-02/ALI) and from the European Commission (Project WINEGAR, Cooperative Research under the Sixth Framework Programme of the European Community, 2005–2007). We also thank the Language Service of the Rovira i Virgili University for revising the manuscript.
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(Guillamón) Current address: Departamento de Biotecnología de los alimentos, Instituto de Agroquímica y Tecnología de Alimentos (CSIC), P.O. Box 73, E-46100 Burjassot, Valencia, Spain.