Acta Univ. Agric. Silvic. Mendelianae Brun. 2010, 58(2), 169-174 | DOI: 10.11118/actaun201058020169

Výběr nejvhodnější metody izolace DNA pro identifikaci druhů z potravin a krmiv

Michaela Nesvadbová, Aleš Knoll, Anna Vašátková
Ústav morfologie, fyziologie a genetiky zvířat, Mendelova univerzita v Brně, Zemědělská 1, 613 00 Brno, Česká republika

V této práci byla izolována DNA z deseti vzorků vybraných potravin a krmiv pomocí osmi různých extrakčních kitů, které jsou běžně dostupné na trhu. Hlavním cílem bylo ověřit kvalitu a kvantitu DNA, vybrat vhodnou metodu pro izolaci DNA z potravin a krmiv a získat spolehlivé výsledky pro další analýzy. Kvalita, koncentrace a čistota izolované DNA byla ověřena pomocí gelové elektroforézy, měření absorbance spektrofotometrem Nanodrop 2000 (Thermo Scientific) a polymerázové řetězové reakce (PCR). DNA extrahovaná z konzervovaných potravin a krmiv byla více degradovaná, její obsah a čistota byla nižší a také PCR analýza byla obtížnější v porovnání s DNA, která byla izolována z masa, šunky, párků, granulovaných krmiv pro psy a kuřecí moučky. NucleoSpin Food a JetQuick Tissue DNA Spin Kit byly vybrány jako nejlepší metody pro extrakci DNA vybraných potravin a krmiv. Extrakce DNA pomocí těchto kitů umožňuje nejvyšší výtěžky DNA, vysokou čistotu a výsledky PCR analýzy mají výbornou reprodukovatelnost.

extrakční metody, potraviny, krmiva, kvalita a kvantita DNA

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

High quality and purity of DNA isolated from food and feed is essential for species identification and has unpredictable influences an effect of analysis. In this study, the efficiency of eight different 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 flour were used. Kits of several different producers, i.e.: NucleoSpin Food (Marchery-Nagel), Wizard Genomic DNA Purification Kit (Promega), Invisorb Spin Food Kit I (Invitek), Wizard SV Genomic DNA Purification 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 verification of DNA quality was performed. The isolates were subsequently assessed for quantity and quality using by spectrophotometer Nanodrop 2000 (Thermo Scientific). To verify of template usability and quality of isolated DNA, the polymerase chain reaction (PCR) was used.
Differences between isolated DNA from tinned products and meat, ham, sausage, granulated dog feed and chicken flour were found. In tinned food and feed, the DNA was more degraded, DNA content and DNA purity was lower and also PCR amplification was the most difficult. Overall DNA yield and quality have important influence on PCR products amplification. 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 different food or feed using and different 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.

Keywords: extraction methods, food, feed, DNA quantity and quality
Grants and funding:

This work was supported by Ministry of Education, Youth and Sports of the Czech Republic by project No. FRVŠ 1305/2009.

Received: January 21, 2010; Published: October 1, 2014  Show citation

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Nesvadbová, M., Knoll, A., & Vašátková, A. (2010). Selection of the most suitable method for the extraction of DNA from foods and feeds for species identification. Acta Universitatis Agriculturae et Silviculturae Mendelianae Brunensis, 58(2), 169-174. doi: 10.11118/actaun201058020169
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