WO2011067630A1 - Method and kit for dna extraction from vitis vinifera l. and for amplification and detection of grapevine varieties or cultivars in musts or wines - Google Patents

Abstract

The present invention describes a method for extracting DNA from Vitis vinifera L. comprising the following stages: a. precipitating the wine or must samples with isopropanol or 2 propanol, extracting and collecting the pellet; b. incubating the sample resulting from the previous stage with an extraction buffer comprising: • preferably a membrane solubilizer; • one or more anti-oxidizing agents; • a chelant of divalent cations, Mg⁺² and/or Ca⁺²; c. performing one or more extractions on the sample resulting from the previous stage using chloroform and/or phenol; d. performing one or more periods of precipitation and incubation with RNases; e. detecting the grapevine varieties or cultivars. The invention also describes a DNA extraction kit for wine or must samples comprising a solution that contains said extraction buffer, and the extraction is conducted according to the described method. The method and kit can be implemented as a traceability system for ascertaining the origin of the product, detecting counterfeits and mislabelling, and represents an advantageous solution in that it allows grapevine DNA extraction from monovarietal, varietal and commercial musts and wines, thereby making the detection and quantification of different grapevine varieties in musts and wines possible.

Description

 Description

 Title of Invention: METHOD AND CASE FOR THE EXTRACTION OF VITIS VINIFERA L. DNA AND FOR THE AMPLIFICATION AND DETECTION OF VARIETIES OF VINE OR CASSAVES IN

MUST OR WINES

 Technical Domain of the Invention

 [1] The present invention falls into two distinct areas, that of its application, in the wine sector, since it covers the whole process of vinification from the vine (leaf and berry), through the wort to the final product, the wine, subsequent bottling and certification, as well as in the area of molecular genetics, where the method of extracting grapevine DNA from monovarietal, varietal and commercial wines and musts and their amplification was developed.

 Introduction

 [2] Viticulture is one of the agricultural activities with the greatest economic impact worldwide. The European Union produces 60% of the world's wine (156 million hL / year), which represents 2.4 billion Euros. However, new wine-producing countries (Chile, USA, South Africa and Australia) are emerging. With the

Portugal in the European Union, the Community nomenclature for wine classification has been adopted and in Portugal, as well as in several countries, the Denominación de Origen means wines whose characteristics and individuality are inseparable from a given geographical region, including natural and human factors. However, for a wine to benefit from a Denomination of Origin, the entire process of wine production is strictly controlled from the vineyard to the consumer, fulfilling a selection of vine varieties authorized for each region, vinification methods and organoleptic characteristics. In Portugal, the body responsible for control, ensuring the genuineness within the demarcated regions (Law no. 8/85, of 4 June) are the Regional Wine Commissions. The first region of Denomination of Controlled Origin (DOC) originated in Portugal in the 19th century. XVIII (Port Wine Region - 1756), however, it is only relatively recently that the demarcation of other regions producing quality wines has become widespread. As mentioned above all control is carried out in wine-growing and wine-growing regions including bottling, labeling and distribution, however, this inspection does not prevent fraud from occurring continuously, especially in certified products with a high economic value, such as Port wine. The fallacious information on labels, in relation to the vine varieties used in the production of a particular wine and the origin thereof, as well as the falsification of certification response by the competent bodies.

 [3] The present invention describes a simple and rapid method of extracting grapevine DNA from wines allowing the detection / quantification of different varieties in musts and wines, thus being of extreme importance in quality control of wine produced , detection of forgeries, increased food safety and rejection of imported wine sold as national product or grapes of different varieties and regions introduced into other regions of DOC wines. SUMMARY OF THE INVENTION

 [4] This invention is a natural consequence of current economic and social demand.

 It comes as a response to the increasingly demanding consumers who require food safety and an accurate verification of the certified product purchased since it allows the detection and consequent quantification of the variety of vine used in the production of a particular wine that is referenced on the label.

 [5] So far, the methods developed for the identification of grapevine varieties did not contain any genetic component, they only comprised protein profiles, anthocyanins, aromatic compounds and chemical elements. In this way, they depended on several factors such as the composition of the soil, climatic conditions, and the processes of vinification and aging of the wine compromising the identification of the grape variety used in the production of a certain must / wine.

 [6] According to existing literature, DNA extraction from grapevine was tested on experimental wines collected immediately after the fermentation process and up to two years of bottling, however, all attempts failed because of the inefficacy of the methods in overcoming the numerous problems encountered in the course of the process. The shortage of DNA obtained at the end of the extraction and its poor quality, the interference of polysaccharides, tannins and polyphenols present in these matrices in DNA amplification prevented the identification of the varieties and led to increasingly complex approaches.

 [7] The method described here does not depend on external factors, it applies to vine DNA in hitherto inaccessible wine samples, is easy to perform, does not comprise dangerous or lethal actions during its implementation, the process is quick methodologies, does not admit of excessive costs, and above all goes beyond all the technical problems set out above. In the end, the results, achieved in an easy and fast way, are accurate (confirm the existence of a certain variety) and unequivocal.

 [8] One of the objects of the present invention is the description of a method of extracting

DNA of Vitis vinifera L. characterized by comprising the following steps:

The. precipitate the wine or must samples with isopropanol or 2-propanol, extract and collecting the sediment;

 B. incubating the resulting sample from the previous step with an extraction buffer comprising:

 a membrane solubilizer, preferably a cationic detergent, CTAB - cetyltrimethyl ammonium bromide;

 one or more anti-oxidant agents;

- a divalent cation chelating agent, Mg ⁺² and / or Ca ⁺² , preferably EDTA

- ethylenediamine tetraacetic acid;

 W. performing one or more extractions of the sample resulting from the previous step using chloroform and / or phenol;

 d. carry out one or more periods of precipitation and incubation with RNases.

 [9] In a preferred embodiment, there is a previous step prior step of precipitating the sample into 2-propanol or isopropanol by centrifuging the sample and collecting the pellet.

 [10] In another preferred embodiment the extraction solution further comprises sodium chloride and tris-HCl. In an even more preferred embodiment the anti-oxidant agents used may be polyvinylpyrrolidone (PVP) and / or β-mercaptoethanol.

 [11] In an even more preferred embodiment the extraction buffer is previously

 heated.

 [12] In a further preferred embodiment the extracted DNA from wine or must samples is further amplified and identified with markers suitable for the variety or caste of Vitis viridis. In an even more preferred embodiment one can amplify and identify the extracted DNA by PCR and with specific markers, of at least one of the following varieties or varieties of Vitis vinifera L. or by hybridization techniques.

 [13] An even more preferred embodiment of the present invention is the identification of DNA extracted by PCR with specific markers from at least one of the following varieties or varieties:

 Alicante Bouschet, Baga, Barbera, Cabernet Sauvignon, Canaiolo, Carménère, Cinsaut, Dolcetto, Gamay, Malbec, Merlot, Mourvèdre, Nebbiolo, Petit Syrah, Pinot Noir, Syrah, Sangiovese, Tannat, TempraniUo or Aragonez, Mole Black Ink , Touriga Nacional, Negra Mole, Blaufrankish or Kekfrancós, Abelhal,

 Alvarinho, Chardonnay, Chenin Blanc, Gewiirztraminer, Malvasia, Muller-Thurgau, Pinot Blanc, Pinot Grigio, Prosecco, Riesling, Sauvignon Blanc, Semillon Blanc, Trebiano, Furmint, Muscat Yellow, Zeta, Bouvier, Riesling, Harslevelu, Milleau, Mosaic Liturcci or Italian Cherry, Riesling Rosè, Saint Tomaint, Terreau.

[14] In an even more preferred embodiment for the amplification and identification of DNA from strains or plant varieties of Vitis vinifera L., the following molecular markers may be used, among others: ssrVVS2, ssrVVMD5, ssrVVMD7, ssrVVMD27, VrZAG62 and ssrVrZAG79.

 In still more preferred embodiment samples of wines or musts to be analyzed may be collected during fermentation, in the final stage of fermentation, in maturation, aging or in bottled wine irrespective of their age.

 [16] In another preferred embodiment the DNA extraction method Vitis vinifera L., in samples of wines or musts the extracted DNA is free from contamination by phenol, polysaccharides, proteins, other phenolic compounds, solvents and salts which absorb 280, 270 and 230 nm.

 [17] In a more preferred embodiment of the method of extracting DNA from Vitis vinifera L., in wine or must samples the obtained DNA contains a number of base pairs comprised between 10 and 2500 bp, preferably between 245 and 249 bp.

 It is further object of the present invention to provide a description of a DNA extraction kit in samples of wine or wort characterized in that it comprises a solution containing the aforementioned extraction buffer, where preferably the extraction of Vitis vinifera L. , in samples of wine or must is performed by the method described above.

 [19] The kit may also contain at least one of the following solutions:

 a precipitation solution which preferably comprises 2-propanol or isopropanol;

 - a washing solution,

 - a solution comprising at least one of the following compounds: phenol, or chloroform, RNase and Proteinase K.

 Background of the Invention

[20] Wine production moves billions of euros per year, thus attracting fraudulent practices. The European Union has adopted specific legislation (178/2002) on food traceability to improve food quality and safety, protect consumers against false product information and thereby promote fair trade. It is therefore essential to associate scientific knowledge with the existing consortium with the aim of developing methods for traceability of wine products so as to guarantee the origin of the product and detect fraud.

 [21] The development of reliable methods for the detection of grapevine varieties in wines is of crucial importance as knowledge of the origin and the protection of controlled denomination of origin products is increasingly a necessity.

[22] To date no method has been Vitis vinifera L. DNA. in wines.

[23] Document ES 2 059 280, of Spanish origin, concerns the method of production of a yeast by recombinant DNA techniques (CETC yeast 1973) and its industrial application as a wine yeast for the improvement of flavorings of the yeast. wine. Thus, the difference in purpose between this patent and the one proposed here is obvious.

 [24] CN 101210032, of Chinese origin, refers to a method of

 preparation of cell cultures (vinification yeasts).

 [25] In the literature, the methods so far developed for the identification of

 varieties of vines or the geographical origin of the grapes comprise protein profiles [Gonzalez-Lara, R., Correa, I., Polo, M.C., Martin-Alvarez, P.J., Ramos, M. (1989). Food Chem. 34: 103-110; Pueyo, E., Dizy, M., Polo, M.C. (1993). Am. J. Enol. Vitic. 44: 255-260; Moreno- Arribas, M.V., Cabello, F., Polo, M.C., Martin-Alvarez, P.J., Pueyo, E. (1999). /. Agne. Food Chem. 47: 114-120], anthocyanins [Revilla, E., Garcia-Beneylez, E., Cabello, F., Martin-Ortega, G., Ryan, J.M. (2001). /.

 Chromatogr. A 915: 53-60; Garcia-Beneytez, E., Cabello, F., Revilla, E. (2003). /. Agne. Food Chem. , 51: 5622-5629], amino acids [Vasconcellos, A.M.P., Chaves das Neves, HJ. (1989). /. Agne. Food Chem. 37: 931-937], aromatic compounds [Munoz-Organero, G. and Ortiz, J.M. (1987) Riv. Vitic. Enol. 3: 55-64] and chemical elements [Almeida, CM. and Vasconcelos, M.T. (2003). /. Agric. Food Chem. 51: 4788-4798; Monaci, F., Bergagli, R. Fcardi, S. (2003). /. Trace Elem. Med. Biol. 17: 45-50; Coetzee, P.P. and Vanhaecke, F. (2005). Anal. Bioanal. Chem. 383: 977-984]. However, these methods have as main drawbacks the duration and dependence of various factors such as soil composition, climatic conditions, vinification processes and aging of the wine, thus compromising the identification of the grape variety (s) used in wine production.

 [26] Vine DNA can be extracted from any part of the plant, however, the

preference lies in young leaves [Lodhi, MA, Ye, GN, Weeden, NF, Reisch, BI (1994). Plant Mol. Biol. Rep. 12: 6-13]. Only a few studies have reported the extraction of DNA from grape juice [Faria, MA, Magalhães, R., Ferreira, MA, Meredith, CP, Monteiro, FF (2000). /. Agric. Food Chem. 48: 1096-1100], must [Baleiras- Couto, MMe Eiras-Dias, JE (2006). Anal. Chim. Acta 563: 283-291; Rodríguez-Plaza, P., González, R., Moreno-Arribas, MV, Polo, MC, Bravo, G., Martínez-Zapater, JM, Martinez, MC; Cifuentes, A. (2006). Eur. Food Res. Technol. 223: 625-631; Faria, M .; Nunes, E .; Oliveira, M. (2008). Eur. Food Res. Technol. 227: 845-850], experimental wines collected immediately after the end of the fermentation process [Baleiras-Couto, MMe Eiras-Dias, JE (2006). Anal. Chim. Acta 563: 283-291; Garcia-Beneytez, E., Maria, VM, Joaquin, B., Maria, CP, Javier, I. (2002). J. Agric. Food Chem. 50: 6090-6096; Siret, R .; Gigaud, O., Rosec, JP, This, P.

 (2002). /. Agric. Food Chem. 50: 3822-3827] and with some age [Savazzini, F. and Martinelli, L. (2006) Anal. Chim. Acta 563: 274-282; Nakamura, S., Haraguchi, K., Mitani, N., Ohtsubo, K. (2007). /. Agric. Food Chem. 55: 10388-10395; Drábek, J .; Stávek, J .; Jal vková, M .; Jurbek, T .; Frébort, I. (2008). Eur. Food Res. Technol. 226: 491- 497], however, none of them presented conclusive results.

 [27] Efficient extraction and DNA amplification of musts and wines is mostly compromised by the interference of polysaccharides, tannins and polyphenols present in these matrices, by biochemical degradation, enzymatic action and the winemaking process itself [Faria, M .; Nunes, E .; Oliveira, M. (2008). Eur. Food Res. Technol. 227: 845-850; Garcia-Beneytez, E., Maria, V.M., Joaquin, B., Maria, C.P., Javier, I. (2002). /. Agric. Food Chem. 50: 6090-6096; Savazzini, F. and Martinelli, L. (2006) Anal. Chim. Acta 563: 274-282; Siret, R .; Gigaud, O., Rosec, J.P., This, P. (2002). /. Agric. Food Chem. 50: 3822-3827]. Previous studies have hypothesized that decantation, clarification, and filtration completely remove DNA from the vine [Garcia-Beneytez, E., Maria, V.M., Joaquin, B., Maria, C.P., Javier, I. (2002). /. Agric. Food Chem. 50: 6090-6096].

 [28] The differentiation of grape varieties by the polymerase chain reaction

 (PCR) using wine as a model is hampered by the scarce amount of DNA present as well as its poor quality. The main causes are degradation of plant DNA during fermentation, contamination by DNAs of microorganisms, in particular yeasts, interference of DNA extraction by polysaccharides and polypeptides, and the coexistence of substances such as polyphenols, which inhibit DNA polymerase in the PCR reaction [Garcia-Beneytez, E., Maria, VM, Joaquin, B., Maria, CP, Javier, I. (2002). /. Agric. Food Chem. 50: 6090-6096; Siret, R .; Gigaud, O., Rosec, J.P., This, P. (2002). /. Agric. Food Chem. 50: 3822-3827]. Recently, new approaches have been adopted to overcome the various difficulties encountered in obtaining DNA extracted from wine and its amplification [Savazzini, F. and Martinelli, L. (2006) Anal. Chim. Acta 563: 274-282; Nakamura, S., Haraguchi, K., Mitani, N., Ohtsubo, K. (2007). /. Agric. Food Chem. 55: 10388-10395; Drábek, J .; Stávek, J .; Jal vková, M .; Jurbek, T .; Frébort, I. (2008). Eur. Food Res. Technol. 226: 491- 497], but none of them have been successful since problems related to the method of DNA extraction and amplification remain.

 [29] With regard to the data published so far published in the literature, the

 the following advantages and problems overcome by the invention described herein:

• Sample type - This invention allows the efficient extraction and consequent amplification of genomic DNA from leaves, wort (beginning of fermentation) and wines, and in this case, considering samples collected at the end of the fermentation and after several years of bottling, something that had not been achieved so far. Some authors tested their protocols in experimental wines collected immediately after the end of the fermentation process [Baleiras-Couto, MM and Eiras-Dias, JE (2006). Anal. Chim. Acta 563: 283-291; and with some age [Savazzini, F. and Martinelli, L. (2006) Anal. Chim. Acta 563: 274-282; Nakamura, S., Haraguchi, K., Mitani, N., Ohtsubo, K. (2007). J. Agric. Food Chem. 55: 10388-10395; Drábek, J., Stávek, J., Jalkov, M., Jurbek, T., Frébort, I. (2008). Eur. Food Res. Technol. 226: 491-497], but never so many years after bottling. In all cases, the results were fruitless.

 • Initial sample volume - The proposed method allows the extraction of grapevine DNA from a volume of wine sample of only 10 mL showing advantageous in relation to the volumes presented in the current literature, ranging from 30 to 400 mL [ Baleiras-Couto, MM and Eiras-Dias, J.E. (2006). Anal. Chim. Minutes 563:

283-291; Savazzini, F. and Martinelli, L. (2006) Anal. Chim. Acta 563: 274-282;

Nakamura, S., Haraguchi, K., Mitani, N., Ohtsubo, K. (2007). /. Agric. Food Chem. 55: 10388-10395; Drábek, J., Stávek, 1, Jal vková, M., Jurbek, T., Frébort, I. (2008). Eur. Food Res. Technol. 226: 491-497].

 • Integrity and quantity of DNA - Previous reports stated that DNA extracted from samples of wine was not always scarce, but always showed poor quality, justifying this mainly with the degradation of DNA during the vinification process [Baleiras-Couto, M.M. and Eiras-Dias, J.E. (2006). Anal. Chim. Acta 563: 283-291; Savazzini, F. and Martinelli, L. (2006) Anal. Chim. Minutes 563:

274-282; Nakamura, S., Haraguchi, K., Mitani, N., Ohtsubo, K. (2007). /. Agric. Food Chem. 55: 10388-10395; Drábek, J., Stávek, J., Jalkov, M., Jurbek, T., Frébort, I. (2008). Eur. Food Res. Technol. 226: 491- 497]. In this study, the quantification of the genomic DNA samples extracted from the wine revealed a high concentration and purity, demonstrating that Vitis vinifera L. DNA remains not only after fermentation, but also resists the whole winemaking process, since the samples of wine were collected at the end of fermentation and up to five years after bottling. In order to solve these problems it was necessary to optimize the DNA extraction protocol of Vitis vinifera L. in wines.

 • Inhibition of the PCR reaction - As mentioned previously, contaminations by polysaccharides, tannins and polyphenols, both in white wines and in red wines [Nakamura, S., Haraguchi, K., Mitani, N., Ohtsubo, K. 2007). /. Agric. Food Chem. 55: 10388-10395], inhibit the PCR reaction. In this method the

combination of the enzymatic treatment, precipitation with isopropanol and the various washes proved the perfect solution for a favorable PCR reaction, eliminating all possible contaminants and allowing to obtain an amplification profile unequivocal.

 • Amplification of DNA - In all tested situations (extraction of DNA from vines in monovarietal and commercial wines, red and white wines) it was possible to amplify the DNA with a specific marker demonstrating that the DNA present corresponded to a variety of grapevines, excluding the hypothesis belonging to a microorganism.

 [30] In this work, a simple primer nuclear primer was used directly on the genomic DNA sample allowing the amplification of the fragment to the correct size. The results obtained showed that the allele sizes found for the wines are in agreement with the sizes found for the vine varieties used in their production. In this way, when using this method, it will be possible to identify the varieties used in the production of a specific wine. This is the first report reporting the amplification of DNA extracted from commercial wine samples. To date, no author has been able to amplify genomic DNA from wine-extracted vine, targeting mitochondrial or chloroplast DNA [Siret, R .; Gigaud, O., Rosec, J.P., This, P. (2002). /. Agric. Food Chem. 50: 3822-3827 Baleiras- Couto, M. M. and Eiras-Dias, J.E. (2006). Anal. Chim. Acta 563: 283-291].

 General Description of the Invention

 [31] The quality, value and price of the wine depends on the vine varieties used, the climatic conditions, the agronomic conditions of the area under cultivation, the vinification technology and the type of storage. Therefore, it is important that consumers rely on the labeling system for the wines used and for this an efficient traceability system must be established.

 [32] In the present invention there is described a simple and efficient method and kit for extracting DNA from vine from wine. Problems such as the quantity and quality of DNA as well as the contaminations with polysaccharides, tannins and polyphenols were totally overcome. The difficulties present in the next phase, corresponding to the PCR amplification, were also overcome and the use of a specific nuclear molecular marker (microsatellite) of vine was sufficient to identify the variety used to produce a certain wine.

 [33] At this time a method of extracting DNA from vine from wine overcoming all the obstacles usually found in this process was established. Thus, since there is no published patent and the articles so far published have not been successful in the experimental methods exposed because they have not been able to prove the existence of grapevine DNA in wine nor its amplification by a simple and effective method , this invention becomes the first in the scope proposed here.

 [34] The importance of this method still lies in the applicability component that

since it can be implemented as a traceability producers, distributors and consumers in order to guarantee the origin of the product, to detect fraud and misleading labeling.

[35] The research team, integrated in the Institute of Bioengineering and Biotechnology - Center of Genetics and Biotechnology of the University of Trás-os-Montes and Alto Douro (IBB-CGB / UTAD) successfully developed a method of extracting genomic DNA from Vitis viniferaL. from single-varietal wines produced in the laboratory and from commercial wines. After obtaining the genomic DNA it was possible to detect the grape variety present in the wines using molecular markers microsatellites.

 [36] Vitis vinifera L. is the most widely cultivated grape vine (Vitis sp.) For wine production in Europe. This vine of the family of grapefruit, whose fruit is the grape, has been cultivated by several European civilizations for thousands of years, which originated dozens of varieties, the so-called grape varieties, through artificial selection.

 [37] The following is a non-exhaustive and indicative list of known varieties or varieties of Vitis vinifera L.:

 Grape varieties: Alicante Bouschet, Baga, Barbera, Cabernet Sauvignon, Canaiolo, Carménère, Cinsaut, Dolcetto, Gamay, Malbec, Merlot, Mourvèdre, Nebbiolo, Petit Syrah, Pinot Noir, Syrah, Sangiovese, Tannat, pranillo or Aragonez, Tinta Negra Mole, Touriga Nacional, Negra Mole,

 Blaufrankish (Kekfrancós in Hungary), among others.

 White Grape Varieties: Abelhal, Alvarinho, Chardonnay, Chenin Blanc, Gewiirztraminer, Malvasia, Muller-Thurgau, Pinot Blanc, Pinot Grigio, Prosecco, Riesling, Sauvignon Blanc, Semillon Blanc, Trebiano, Furmint, Yellow Muscat, Zeta , Bouvier, Riesling, Harslevelu, among others;

 Grape Varieties: Milleau, Mosaic Liturcci or Italian Cherry, Riesling Rosè, Saint Tomaint, Terreau, among others.

 [38] However, it should be noted that there are more than 16000 varieties of varieties and varieties, of which only more than 450 are known in Portugal. It is also worth mentioning that new varieties are always emerging as a result of plant breeding, to meet the increasingly demanding demands of the wine field.

 [39] The DNA extraction and amplification method detects Vitis vinifera L. in wine samples comprising the following steps:

 - The protocol begins with the collection of a sample of wine or must, preferably of only 10 mL;

 The sample is precipitated, preferably 2-propanol or isopropanol, centrifuged and the supernatant is collected;

- The sample is then incubated with an extraction buffer containing: • a CTAB cetyl detergent (cetyltrimethyl ammonium bromide) which solubilizes the membranes,

 • anti-oxidant agents, preferably PVP (polyvinylpyrrolidone) and / or β-mercaptoethanol,

• a divalent cation chelating agent, Mg ⁺² and Ca ⁺² , preferably EDTA (ethylenediamine tetra acetic acid) inhibiting the action of the DNAs and with low concentrations of sodium chloride which favors together with the CTAB the precipitation of DNA ,

 - In separate phases, several extractions are carried out using phenol and / or chloroform, which denature the proteins making them insoluble in the aqueous phase, where the nucleic acids are, thus allowing the separation between these components,

 - The protocol also includes two periods of precipitation at -20 ° C and incubation with RNases, the latter to eliminate RNA (ribonucleic acid) from the samples.

 [40] At the end, the DNA of the vine is obtained, which is then quantified and

 used in polymerase chain reaction (PCR) amplification with specific markers (microsatellites) to identify or determine the various varieties / varieties present in the wine / must sample. It may be used with other types of PCR markers or in a hybridization technique such as microarrays.

 [41] With this protocol problems such as the amount and integrity of DNA have been overcome as well as all the previously described contaminations that inhibit the PCR reaction, obtaining in the end reliable and unambiguous results.

 Description of Figures

 Figure 1 represents the UV spectrum obtained by the NanoDrop® ND-1000 spectrophotometer revealing (A) presence of phenol contamination and (B) absence of contamination by phenol and / or other substances in wine DNA samples.

 [43] Figure 2 represents the amplification profile obtained with the nuclear primer

 VrZAG79. (A) 1/2/3 - white commercial wines, 4 - white monovarietal wine, 5 - 'Fernão Pires' variety (leaf); (B) 1/2/3 - red commercial wines, 4 - red monovarietal wine, 5 - 'Tinta Roriz' variety (leaf); M - molecular marker GeneRuler ™ 100 bp DNA Ladder (Ferments).

 Detailed Description of the Invention

 [44] The extraction method comprises the following steps:

 1 - Precipitate 10 mL of wine (or must) in 2-propanol (70% - 100%) in a centrifuge tube; homogenize and place at -20 ° C for a minimum of one to two weeks, and the precipitation period may extend;

2 - After precipitation, centrifugation at 2500 (2500-4000) rpm for 30 (10 to 45) minutes at room temperature;

 3 - Remove the supernatant;

 4 - Add to the precipitate 0.750 (0.750 to 1) mL extraction buffer [20 mM EDTA (ethylenediamine tetraacetic acid), 100 mM Tris-HCl (hydroxymethyl amino methane hydrogen chloride, pH 8), 1.4 M NaCl Sodium chloride), 2 (1 to 4)% (w / v) CTAB (cetyltrimethyl ammonium bromide)] and to which 2 (1 to 4)% (w / v) of polyvinylpyrrolidone (PVP) and 1 , 5 to 2)% of β-mercaptoethanol; the buffer was preheated to 65 (50 to 70) ° C; homogenize by inversion and add 100 μL of proteinase K (20 mg / mL);

 5 - Incubate at 65 (50 to 70) ° C for a minimum of 1 h, homogenizing at approximately every 15 minutes;

 6 - Add 750 (1 volume) chloroform-isoamyl alcohol mixture (24: 1); homogenize the samples for approximately 10 minutes;

 7 - Centrifuge at 13,000 (maximum rotation) rpm for at least 15 minutes, and transfer the supernatant carefully to another tube (do not disturb the interface);

8 - Treat the samples for a minimum of 1 h at room temperature, with 1-4 μm RNase (Ribonuclease A) (10 mg / mL) or approximately 30 minutes at 37 ° C;

9 - Precipitate the DNA with 0.6 volumes of isopropanol at -20 ° C overnight, the precipitation step being prolonged for several days;

 10 - Centrifuge at 3000 (2500-4000) rpm for at least 15 minutes at 4 (0 to 10) ° C. Neglect the supernatant;

 11 - Resuspend the pellet in 300 to 1000 TE (Tris-EDTA) IX;

 Add 300 to 1000 phenol and homogenize for approximately 10 minutes;

 13. Centrifuge at 8,000 (7,000 to 10,000) rpm for about 15 minutes at 4 (0 to 10) ° C. Transfer the aqueous phase to another tube;

 14 - Repeat steps 11 and 12, as many times as necessary up to a maximum of 5.

 15 - Re-test the samples for a minimum of 1 h at room temperature, with 1-4 μE / mL RNase (10 mg / mL) or approximately 30 minutes at 37 ° C;

 Precipitate the DNA with 0.6 volumes of cold isopropanol at -20 to -25 ° C overnight, the precipitation step being prolonged for several days;

 17 - Centrifuge at 3,000 (2,500-4,000 rpm) for a minimum of 15 minutes at 4 (0 to 10) ° C. Neglect the supernatant;

 Add 0.750 (0.250 to 1.5) mL wash buffer (76% ethanol, 10 mM ammonium acetate) and homogenize for at least 5 minutes;

 19 - Centrifuge at 3,000 (2,500-4,000 rpm) for a minimum of 10 minutes. Remove the supernatant;

20 - Allow the sediment to dry; 21 - Dilute the DNA in 30 (10 to 100) TEO.lx;

 22 - Quantify and store at -20 to -80 ° C.

[45] Examples

 For a more complete understanding of the invention, examples of preferred embodiments of the invention will be described below, which, however, are not intended to limit the subject matter of the present invention.

 [46] Example 1 - DNA extraction and comparison of methodologies - A consistent and reliable DNA extraction method is the basis for the determination of the varietal composition of a particular wine through molecular markers. Thus, 5 previously described DNA extraction methods were tested and compared, a commercial kit with the protocol described herein (Table 1). The method developed proved to be advantageous over the remainder since not only did a higher DNA concentration but also a higher yield were obtained in addition to the initiation of the lower sample volume protocol.

 [47] Table 1. Comparison between seven different DNA extraction methods, considering the initial volume of wine, the mean of the DNA concentration and the total yield

 [Table 1]

 [Table]

[48] Example 2 - Quantification and purity of DNA samples - Phenol is often used in DNA extraction methods increasing the risk of contamination. This reagent shows the maximum absorbance between 270-275 nm, near the wavelength of absorbance of the DNA which sometimes conditions the results since it raises the values of both the purity and the concentration of DNA due to an overvalued reading of the obtained value with the absorbance of 260 nm (A 26o) - Nas Other contaminants such as polysaccharides, proteins, other phenolic compounds, solvents and salts absorb at 280, 270 and 230 nm. Again, the protocol described herein avoids these problems, as demonstrated by Figure 1. This shows the UV spectra obtained by the NanoDrop spectrophotometer on two samples of genomic DNA extracted from wine: in sample A, in which another method was used, reveals phenol contamination and sample B, in which the method described here was used, which revealed absence of

 phenol contamination. The spectrum further shows the absence of any other contamination of the above DNA sample.

 [49] The readings performed on the NanoDrop® ND-1000 spectrophotometer revealed a high concentration of genomic DNA obtained from leaves, monovarietal and commercial wines (Table 2). Concentrations in commercial wines ranged from 164 ng / μL · (white) to 452 ng / μΐ. (red) and the degree of purity presented values between 1.71 and 1.81. These values are lower than those obtained for the leaves, however, they are excellent results for the wines.

[50] Table 2. Determination of the concentration and degree of purity of genomic DNA obtained from monovarietal and commercial leaves and wines

[Table 2]

 [Table]

 [51] Example 3 - Amplification of genomic DNA obtained from wines - After obtaining good quality and quantity DNA, amplification appears as the next step. The PCR reaction can be inhibited by several factors. In wine samples and among several factors, the presence of phenolic compounds and

 polysaccharides are the main cause for the amplification deadlock. However, these contaminants were eliminated and the amplification was successful.

[52] The European project GENRES-081 (http://www.genres.de/idb/vitis/vitis.htm) established a set of six molecular markers (ssrVVS2, ssrVVMD5, ssrVVMD7, ssrVVMD27, VrZAG62 and ssrVrZAG79) of the type microsatellites (SSR) as universal markers for grapevine genotyping. Thus, after extraction of the must and / or wine DNA, these markers allow the identification of specific genotypes of grape varieties and consequently the detection of their involvement in the production of a particular wine. Another objective was to demonstrate that the extraction method developed was efficient and that the genomic DNA sample extracted from wine was suitable for amplification. For this purpose, only the first nuclear primer VrZAG79 [Sefc, KM, Regner, F., Turetschek, E., Glössl, JH, Steinkellner, H. (1999). Genome 42: 367-373] for the differentiation of varieties of vine. The amplification was consistent and visible on an agarose gel (Figure 2) and it was possible to identify the vine varieties used in monovarietal and commercial wines (red and white) with a nuclear marker. To date, other authors have reported only the amplification of DNA from experimental wine samples collected immediately after fermentation [Garcia-Beneytez, E., Maria, VM, Joaquin, B., Maria, CP, Javier, I. (2002) . /. Agric. Food Chem. 50: 6090-6096; Siret, R., Gigaud, O., Rosec, JP, This, P. (2002). /. Agric. Food Chem. 50: 3822-3827].

 [53] The sizes of the alleles obtained with the microsatellite VrZAG79 obtained in the automatic sequencer and expressed in base pairs are shown in Table 3. The size of the alleles in monovarietal wines (white and red) is in agreement with the values found to the leaves. For commercial wines a range of values has been found which comprise all alleles of the varieties used in the production of those wines.

[54] Table 3. Genotypes obtained using the nuclear primer VrZAG79 expressed in allele size and range values found in base pairs, obtained from leaf genomic DNA, monovarietal and commercial wines.

[Table 3]

[Table]

 In this study, only the commercial wines of the Douro region were used, having as reference two varieties (leaves) widely used in wine production in this region. However, the method described here was also successful for a commercial red wine, Bordeaux (2006), where the Merlot and Cabernet Sauvignon varieties were recognized worldwide (results not shown).

Claims

 Claims

 Method of extracting DNA from Vitis vinifera L. characterized by comprising the following steps:

The. precipitate from the wine or must samples with isopropanol, extract and collect from the sediment;

B. incubating the resulting sample from the previous step with an extraction buffer comprising:

W. incubating the wine or must sample with an extraction buffer comprising:

 a membrane solubilizer, namely a cationic detergent, CTAB - cetyltrimethyl ammonium bromide;

 one or more anti-oxidant agents;

- a divalent cation chelating agent, Mg ⁺² and / or Ca ⁺² , namely EDTA

 - ethylenediamine tetraacetic acid;

d. performing one or more extractions of the sample resulting from the previous step using chloroform and / or phenol;

and. carry out one or more periods of precipitation and incubation with RNases.

 A method according to the preceding claim characterized in that the membrane solubilizer is the cationic detergent CTAB - cetyltrimethyl ammonium bromide.

A method according to any one of the preceding claims characterized in that the divalent cation chelating agent, Mg ⁺² and / or Ca ⁺² , is EDTA - ethylenediamine tetraacetic acid.

 A method according to any one of the preceding claims characterized in that the extraction buffer further comprises sodium chloride and tris-HCl.

 A method according to any one of the preceding claims characterized in that the anti-oxidizing agents are polyvinylpyrrolidone (PVP) and / or β-mercaptoethanol.

 A method according to any one of the preceding claims characterized in that it comprises the previous step of precipitating the sample into 2-propanol or isopropanol, centrifuging and collecting the pellet.

A method according to any one of the preceding claims, characterized in that the extraction plug is preheated.

 A method according to any one of the preceding claims characterized in that the extracted DNA is subsequently amplified and identified with labels suitable for the Vitis vinifera L. variety or caste.

 Method according to the previous claims characterized to identify the extracted DNA by hybridization techniques.

A method according to any one of the preceding claims characterized in that the amplified and identified DNA is extracted by PCR and with specific markers of at least one of the following varieties or varieties:

 Alicante Bouschet, Baga, Barbera, Cabernet Franc, Cabernet

Sauvignon, Canaiolo, Carménère, Cinsaut, Dolcetto, Gamay, Malbec, Merlot, Mourvèdre, Nebbiolo, Petit Syrah, Pinot Noir, Syrah,

Sangiovese, Tannat, Tempranillo or Aragonez, Tinta Negra Mole, Touriga Nacional, Negra Mole, Blaufrankish or Kekfrancos, Abelhal, Alvarinho, Chardonnay, Chenin Blanc, Gewiirztraminer, Malvasia, Muller-Thurgau, Pinot Blanc, Pinot Grigio, Prosecco, Riesling, Sauvignon Blanc, Semillon Blanc, Trebiano, Furmint, Muscat Yellow, Zeta, Bouvier, Riesling, Harslevelu, Milleau, Mosaic Liturcci or Italian Cherry, Riesling Rosè, Saint Tomaint, Terreau.

 A method according to the preceding claims characterized by amplifying and identifying the extracted DNA by PCR using at least one of the following molecular markers ssrVVS2, ssrVVMD5, ssrVVMD7, ssrVVMD27, VrZAG62 and

 ssrVrZAG79.

 Method according to any one of the preceding claims characterized in that samples of wine or must, collected during fermentation, at the final stage of fermentation, at maturation, aging or bottled wine, irrespective of their age, are used.

 Method according to any one of the preceding claims characterized in that the DNA is free of phenol, polysaccharides, proteins, other phenolic compounds, solvents and salts which absorb at 280, 270 and 230 nm.

Method of extracting and amplifying DNA according to any one of the preceding claims characterized in that DNA containing a number of base pairs is comprised between 10 and 2500 bp, preferably between 245 and 249 bp.

 [Claim 15] A DNA extraction kit in wine or wort samples which comprises a solution containing the extraction buffer of any one of claims 1-14.

A DNA extraction kit in wine or must samples according to the preceding claim, characterized in that it comprises said extraction buffer and the remaining precursors according to the method of any one of claims 1-14.

Claim 17 The kit according to any one of claims 15-16, characterized in that it comprises at least one of the following solutions:

- a precipitation solution which preferably comprises

 2-propanol or isopropanol;

 - a washing solution,

 - a solution comprising at least one of the following compounds: phenol, or chloroform, RNase and Proteinase K.