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NESVADBOVÁ, M., KNOLL, A., VAŠÁTKOVÁ, A.: Selection of the most suitable method for the extraction of DNA from foods and feeds for species identifi cation. Acta univ. agric. et silvic. Mendel. Brun., 2010, LVIII, No. 2, pp. 169–174 High quality and purity of DNA isolated from food and feed is essential for species identifi cation and has unpredictable infl uences an eff ect of analysis. In this study, the effi ciency of eight diff erent methods for DNA isolation was investigated. For DNA extraction, the raw chicken meet, ham, sausages, tinned lunch meat, pate, tinned feeds for dogs, complete granulated feeds for dogs and chicken fl our were used. Kits of several diff erent producers, i.e.: NucleoSpin Food (Marchery-Nagel), Wizard Genomic DNA Purifi cation Kit (Promega), Invisorb Spin Food Kit I (Invitek), Wizard SV Genomic DNA Purifi cation System (Promega), JetQuick Tissue DNA Spin Kit (Genomed), RNA Blue (Top-Bio), JetQuick Blood & Cell Culture Kit (Genomed), QIAamp DNA Mini Kit and QIAamp DNA Blood Mini Kit (Qiagen) were employed in the study. Gel agarose electrophoresis for primary verifi cation of DNA quality was performed. The isolates were subsequently assessed for quantity and quality using by spectrophotometer Nanodrop 2000 (Thermo Scientifi c). To verify of template usability and quality of isolated DNA, the polymerase chain reaction (PCR) was used. Diff erences between isolated DNA from tinned products and meat, ham, sausage, granulated dog feed and chicken fl our were found. In tinned food and feed, the DNA was more degraded, DNA content and DNA purity was lower and also PCR amplifi cation was the most diffi cult. Overall DNA yield and quality have important infl uence on PCR products amplifi cation. The best results were obtained with NucleoSpin Food and JetQuick Tissue DNA Spin Kit. DNA extracted by these methods proved highest yields, purity and template quality in all foods and feeds and the results of PCR analysis are excellent reproducible. Analyses showed that results depended on diff erent food or feed using and diff erent isolation system. The results of this work will be utilized to choose the suitable isolating kit for educational course, which is designed for students and also for following research and analyses. extraction methods, food, feed, DNA quantity and quality Species identifi cation of animal tissues in meat products is very important for consumers and pet breeders. Food and feed products must be exactly labelled as to the species they contain. Con su mers need high quality products that are labelled correctly in order to assure meat safety (Weibin et al., 2009). Nowadays, various methods for diff e rentia tion and identifi cation of meat products are described and discussed. The analyses using nucleic acid have shown to be most appropriate to reveal species in food and feed (Calvo et al., 2001; Girish et al., 2004). The DNA molecule is more thermostable than many proteins and these molecules are present in majority of the cells of organisms (Lockley and Bardsley, 2000). Nevertheless, the basic problem of analysis and detection of animal species in foods and feeds is quantity and quality of the extracted DNA. The DNA molecule suff ers degradation due to thermal treatments to which foods and feeds are subjected during the production process such as cooking, sterilisation and extrusion (Saez et al., 2004; Engel et al., 2006; Aslan et al., 2009). Also 170 M. Nesvadbová, A. Knoll, A. Vašátková the type of liquid added to the product may play a role in the degradation of DNA (Bauer et al., 2003; Chapela et al., 2007). DNA quality, quantity and purity do have substantial eff ect upon the species identifi cation, therefore the methods for extracting DNA should be carefully selected (Sagi et al., 2009). The aim of the present study was: 1) to analyse the ability of diff erent commercial methods to extract DNA from several food and pet food samples, 2) to evaluate the quantity and quality of extracted DNA, 3) to select the best extraction protocol for a food and feed samples and 4) to obtain dependable results of isolation for further analyses. The kits were chosen on the basis of their avai labi li ty in the market, time demands of the particular procedure, price per individual isolation, to xici ty of the kit chemicals and the extraction method (column fi lter or phenol-chloroform extraction). Methodology of reagents testing should belong to the standard practice of the laboratory. The present results of this work form the part of educational course, which is designed for students of Mendel University in Brno. MATERIAL AND METHODS Ten diff erent samples that contained chicken meat were studied: raw meat, ham, sausage, tinned lunch meat, pate, tinned feed for dogs (two samples), complete granulated feed for dogs (two samples) and chicken fl our. The samples were homogenized using mixer and DNA was isolated following these extraction techniques according to manufacture’s protocols: NucleoSpin Food (Marchery-Nagel), Wizard Genomic DNA Purifi cation Kit (Promega), Invisorb Spin Food Kit I (Invitek), Wizard SV Genomic DNA Purifi cation System (Promega), JetQuick Tissue DNA Spin Kit (Genomed), RNA Blue (Top-Bio), JetQuick Blood & Cell Culture Kit (Genomed) and QIAamp DNA Mini and Blood Mini (Qiagen). Tab. I shows further important characteristic of each kit. The DNA concentration and purity was determined by measuring absorbance by spec tro photo me ter NanoDrop 2000 (Thermo Scientifi c). Methods to evaluate the quality of the extracted DNA were electrophoresis on 1% agarose gel and po lyme ra se chain reaction (PCR). The primers to amplify the chicken-specifi c PCR product were assumed according to Matsunaga et al. (1999). The size of the obtained PCR product was 227 bp. PCR reactions were performed in total volumes 25 μl containing 200 μM of each dNTP, 0.2 μM of each primer, 1 Units of LA DNA polymerase in standard PCR buff er (Top-Bio, CZ) and 2 μl of DNA template. Amplifi cation was performed in a thermocykler PTC 200 (MJ Research) and the cycling conditions were initial denaturation at 95°C (2min), followed by 35 cycles of 95 °C (30s), 52 °C (30s), 68 °C (30s), with fi nal extension at 68 °C (7min). PCR products were checked by electrophoresis in 3% agarose gel using TBE buff er with ethidium bromide (0.5 μg/ml) staining. RESULTS AND DISCUSSION The DNA from ten diff erent samples of foods and feeds, containing chicken meat, was analyzed. DNA was extracted by eight isolation methods. The aim of extraction procedures was to obtain high quality and quantity DNA suitable for for species identifi cation. The methods agarose gel electrophoresis, measuring absorbance by spectrophotometer and PCR were performed. The results of all analysis were in the correlation and corresponded with others authors, as it is demonstrated later. The DNA extraction methods have impact on quantity and quality of the extracted DNA and on the effi ciency of the DNA amplifi cation therefore (Di Bernardo et al., 2007). Exposure to heat and physical or chemical treatments are known to cause fragmentation of DNA molecules and random breaks in DNA strands (Quinteiro et al., 1998; Peano et al., 2004). The method of the extraction, the type of liquid employed for canning, cooking and other processing of meet lead to degradation of DNA and can have a great infl uence on DNA quality, quantity and results in PCR analyses (Chapela et al., 2007; Aslan et al., 2009). DNA quality is a critical factor for most amplifi cation-based analyses, because the amplifi cation of DNA is infl uenced by the presence of inhibitors from the matrix or the extraction reagents, which can reduce the effi ciency of the PCR reaction (Smith et al., 2005). Nevertheless, DNA which underwent thermal denaturation can be still detected General description of DNA extraction methods I: Method Base of system Amount of starting material (mg) Elution time (min) volume (μl) number NucleoSpin column 200 5 100 1 Wizard kit pellet 20 60 100 1 Invisorb column 40 3 400 2 Wizard SV column 20 2 250 2 JetQuick tis. column 20 5 200 2 RNA Blue pellet 100 300 1 JetQuick bl. column 20 2 200 2 QIAamp column 20 5 200 2 Selection of the most suitable method for the extraction of DNA from foods and feeds for species identifi cation 171 by short fragment amplifi cation (Krcmar, Rencova, 2001) but the length of PCR amplicon is cardinal aspect for detection of DNA which was denatured by cooking and others thermal processing (Meyer et al., 1994; Hird et al., 2006; Martín et al., 2008). The correct choice of the DNA extraction method and DNA quantifi cation are very important steps in the analytical procedure to ensure optimal results (Barbaro et al., 2004). Gel electrophoresis showed possibility to detect the band corresponding to the genomic DNA in meat, ham, sausage, granulated dog feed and chicken fl our. DNA isolated from tinned lunch meat, pate and tinned feed for dogs, was substantially da ma ged (Fig. 1). The spectrophotometric measurements (Tab. II and Tab. III) also proved that DNA yield of meat, ham, sausage, granulated dog feed and chicken fl our was higher (the average DNA concentration in isolates obtained by all extraction method was 126.4 ng / μl) than of those from tinned samples (the average DNA concentration in isolates obtained by all extraction method was 33.4 ng/μl). Additionally, DNA quality which is determined by the A260/A280 ratio diff ered between these samples groups (average 1.75 and 1.66, respectively). All eight isolation procedures gave good results in DNA concentration and quality measurement in presence of chicken meat, ham, sausage, granulated dog feed and fl our. The highest DNA yields were obtained by NucleoSpin Food, Wizard Genomic DNA Purifi cation Kit and JetQuick Tissue DNA Spin Kit (however, this also depends on diff erent factors such as input amount of samples, elution time, temperature of elution buff er, fi nal elution volume, number

Selection of the most suitable method for the extraction of DNA from foods and feeds for species identification