Food Control 18 (2007) 76–80 www.elsevier.

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A comparison of DNA extraction methods for food analysis
Angela Di Pinto a,*, VitoTony Forte a, Maria Corsignano Guastadisegni b, Carmela Martino b, Francesco Paolo Schena b, Giuseppina Tantillo a
a

` e Benessere degli Animali, Universita ` degli Studi Bari, Facolta ` di Medicina Veterinaria, Provinciale per Casamassima, km 3, Dipartimento di Sanita 70010 Valenzano-Bari, Italy b Apuliabiotech S.C.R.L., Provinciale per Casamassima, km 3, 70010 Valenzano-Bari, Italy Received 31 January 2005; received in revised form 11 August 2005; accepted 12 August 2005

Abstract In this paper, two DNA extraction and purification procedures for food analysis, WizardÒ Magnetic DNA Purification for Food (Promega) and DNeasyÒ Tissue Kit (QIAGEN), have been compared concerning extraction efficiency, DNA purity and DNA suitability for amplification. The comparison of two extraction methods, in this study, has highlighted the efficiency of the WizardÒ Magnetic DNA Purification approach for vegetable matrices while the revised DNeasyÒ Tissue Kit for complex and processed matrices. Ó 2005 Elsevier Ltd. All rights reserved.
Keywords: Food; DNA extraction; DNA purification; PCR

1. Introduction In the last years several molecular methods have been proposed as useful tools for the food analysis (Bottero, Civera, Anastasio, Turi, & Rosati, 2002; Calvo, Zaragoza, & Osta, 2001; Lipp et al., 2001; Lockley & Bardsley, 2000; Pan, 2002; Soule et al., 2000). Although the DNA-based methods, such as polymerase chain reaction (PCR), are highly specific, reproducible, sensitive and characterized by high discriminatory power, rapid processing time and low costs, they are strongly limited by the presence of inhibitors in food. The PCR inhibitors, such as polysaccharides, humic acids, particularly abundant in food samples are not completely removed during classical extraction protocols remaining as contaminants in the final DNA preparations. The inhibitor compounds can interfere with the reaction at several levels, leading to decreasing and even to complete inhibition of DNA polymerase activity.

*

Corresponding author. Tel.: +390805443970; fax: +390805443855. E-mail address: a.dipinto@veterinaria.uniba.it (A.D. Pinto).

Thus, the DNA-based methods are highly dependent on the DNA extraction and purification techniques. In particular, the application of molecular methods to food samples requires stringent extraction and purification strategies that ensure efficient recovery of nucleic acid and removal of the numerous compounds inhibiting PCR assay (Lipp et al., 2001; Meyer, 1999; Meyer & Candrian, 1996; Zimmermann, Luthy, & Pauli, 1998). The methods reported for extraction and purification of nucleic acids from different food samples involve often organic extraction and ethanol precipitation with a variable loss of non-negligible amounts of the original sample (Arnal et al., 1999; Atmar, Metcalf, Neil, & Esters, 1993; Lewis & Metcalf, 1988; Schwab, Neill, Le Guyader, Estes, & Atmar, 2001). In this paper, two DNA extraction and purification procedures for food analysis, WizardÒ Magnetic DNA Purification for Food (Promega Italia S.r.l., Milano, Italy) and DNeasyÒ Tissue Kit (QIAGEN, Hilden, Germany), have been compared concerning extraction efficiency, DNA purity and DNA suitability for amplification. Although both approaches involve the extraction of DNA using the silica as affinity matrix, one procedure uses a mobile solid phase, while the other is a column-based system. The comparison

0956-7135/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodcont.2005.08.011

6 100. 2. Italy).2 8–10 A260/280 range 1.8 15. Pinto et al. The tube was mixed and incubated at room temperature for 5 min by shaking.2 28..0 62.0 1.4–69. from horse and cow (BIOTOOLS B&M Labs.A. the liquid phase was discarded.r.1 PCR LOD Spike test IRMM-411R-4 2% Bt-176 Maize flour Poultry feed Chocolate cream Mays oil WizardÒ Magnetic DNA DNeasyÒ Tissue kit WizardÒ Magnetic DNA DNeasyÒ Tissue kit WizardÒ Magnetic DNA DNeasyÒ Tissue kit WizardÒ Magnetic DNA DNeasyÒ Tissue kit WizardÒ Magnetic DNA DNeasyÒ Tissue kit WizardÒ Magnetic DNA DNeasyÒ Tissue kit WizardÒ Magnetic DNA DNeasyÒ Tissue kit WizardÒ Magnetic DNA DNeasyÒ Tissue kit WizardÒ Magnetic DNA DNeasyÒ Tissue kit WizardÒ Magnetic DNA DNeasyÒ Tissue kit WizardÒ Magnetic DNA DNeasyÒ Tissue kit WizardÒ Magnetic DNA DNeasyÒ Tissue kit Cherry marmalade Potato dumpling Chocolate snack Vegetable bouillon cube Bran Horse meat Pasteurised milk Purification Purification Purification Purification Purification Purification Purification 0.1.1 50.25–2.0 0 328.3–56.7 2–4.0 0–0.85–2.1 1.9 3. The final volume was adjusted to 100 ll by adding nuclease-free water.6 0 0 20.000g.5 14. Table 1 Results Sample (200 mg) Extraction methods DNA concentration range (ng/ll) Purification Purification Purification Purification Purification 98. 2.D. The tube was placed onto the MagneSphereÒ Magnetic Separation Stand (Promega Italia S.. DNA extraction by WizardÒ magnetic DNA purification for food Two hundred milligram of sample were vortexed vigorously with 500 ll of lysis buffer A and 5 ll of RNase A. Italy) and left in place for 1 min.97 0. After 1 min incubation at room temperature. Italy) and DNeasyÒ Tissue Kit (QIAGEN.46–1.4 46.3 0.4 90.2 59.05 ng/ll 2. Then.7–14. Belgium). Madrid. Milano.r.5 pg/ll 2.59–1. 2.8 102.04 0 1.2–141. 100 ll of nuclease-free water was added to particles and the mixture obtained was mixed and incubated at 65 °C for 5 min.6 98. Geel.2 9.70–1. Milano.43 1. Spain) were used as positive controls.2 40.4 0 0 26. Then the tube was placed onto the MagneSphereÒ Magnetic Separation Stand (Promega Italia S.3 0.7–38.5 pg ng/ll 0. The tube was mixed and replaced on MagneSphereÒ Magnetic Separation Stand (Promega Italia S.4 1.47 1.5 67. after 1 min in the magnetic stand.2 0.67 1.1–0. 1 ml of 70% ethanol wash solution was added and.11 1.76–1. the liquid phase was discarded.05 ng/ll 0. Italy) for 1 min and the DNA was collected by leaving the tube in the stand and carefully transferring the liquid into a clean tube.r.8–467. The liquid phase was discarded leaving the tubes in the stand.89–1.08 0.06 0 0 0.5 pg/ll 0.75–1.8–20..9–80.l.2 38.3–37.125 pg/ll 2.62–1.5 pg/ll Not Not Not Not Not Not – – + Not Not Not – Not – Not Not Not Not Not – Not Not Not tested tested tested tested tested tested tested tested tested tested tested tested tested tested tested tested tested tested .5 pg/ll 0. was added to 750 ll of precipitation solution.37 1.3–52.40–1.r. This step was repeated for three times and in the end the particles were dried at room temperature for 15–30 min.8 3.3. Milano.3 139. The supernatant was added to 50 ll of resuspended MagneSilTM PMPs and 1 ml of isopropanol was added.125 pg/ll ND 0.5 11.7 12. / Food Control 18 (2007) 76–80 77 of extraction and purification procedures was performed from food and feed samples. Milano.77 DNA concentration media 60. Then sample.5 125. Positive controls DNA from Certified Reference Material IRMM-411R-4 2% Bt-176 maize (Fluka.8–70 96. Germany).3 48.0–1.0–50 0–12.3.39 0.56–2.l. The tube was removed from the stand and 250 ll of lysis buffer B was added to the particles.95–1.5 26.88–1.5–128. incubated for 10 min at room temperature with 250 ll of Buffer B.3 0 376.2 28.2–64.7 1.05 ng/ll ND ND ND Amplicon detected with 4 ll of template 0.2–44.6–19.00 1.1 9.05 ng/ll ND 2.11–1.05–2.l.2 37.6 13.0–8. DNA extraction DNA was extracted and purified using two commercial kits based on use of silica as affinity matrix: WizardÒ Magnetic DNA Purification for Food (Promega Italia S.5 pg/ll 1 ng/ll 0.6 8.13–1.37–1. Hilden. 2.5 11.2.4 106. Samples Food and feed samples listed in Table 1 were used in this study.1 38. Material and methods 2.1.05 ng/ll ND 2..99–1.8–93. The mixture obtained was centrifuged for 10 min at 13.l.5 pg/ll 1 ng/ll 2.78 0.8 48.125 pg/ll 2.

carried out in a Mastercycler 5332 (Eppendorf.78 A. 2. Germany). Determination of DNA concentration and purity DNA concentration was determined by measuring the absorbance at 260 nm. upstream primer 5 0 ggc tta tat tac ggg tct tac act-3 0 and downstream primer 5 0 - ggc aat tgc tat gat gat aaa tgg a-3 0 (Bottero et al.5.89 M Tris.1 and 1. pH 8. extension at 72 °C of 30 s and a final extension at 72 °C for 7 min.2. The PCR reactions which gave no amplicons were subjected to spiked controls for identification the presence of PCR inhibitors. DNA purity was measured by calculating the ratio of absorbance at 260–280 nm.). The reaction was performed in a final volume of 25 ll using 12. The cycling conditions for the PCR assay were the same of the program used for Bovine milk samples. Vegetable samples PCR test of vegetable samples was carried out using primer pair. Germany). Vilnius. 3. 2002). The cycling conditions for the PCR assays. Moreover. Lithuania) was used as marker. Sweden) gel in 1· TBE buffer containing 0. Italy). Gielly. Hilden. Italy) with an initial denaturation step of 95 °C for 15 min. gave the results reported in Table 1 and in Figs. Then DNA bound to the QIAamp membrane was washed by centrifugation using two different washing buffers.3. 0. The purified DNA was eluted from the QIAamp spin column in 100 ll of elution buffer. Milan. Germany).5. 0.1. The reaction was performed in a final volume of 25 ll using 12.5 lM of each primer and 1 ll of serial dilution of DNA from 1 lg/ll to 6 fg/ll. was added to 3 ml ethanol. 3.1. the volume of Proteinase K and lysis buffers and the number of washing steps. A spectrophotometer BioPhotometer (Eppendorf. were: denaturation step of 95 °C for 15 min.4. 0. carried out by measuring the DNA concentration and suitability for PCR. DNA extraction by DNeasyÒ Tissue Kit The protocol for purification of total DNA from animal tissue was revised concerning the quantification of starting material. A Gene RulerTM 100 bp DNA Ladder Plus (MBI Fermentas. were targeted a 279 bp fragment of mitochondrial cytochrome b gene of bovine specie.5% (w/v) agarose NA (Pharmacia. annealing at 58 °C for 30 s and extension at 72 °C for 30 s and a final extension at 72 °C for 7 min. The PCR was processed in a Mastercycler 5332 (Eppendorf.97.D. The supernatant. 200 mg of sample were homogenized with 3 ml of lysis buffer ATL and 100 ll proteinase K (20 mg/ml). In particular. were targeted a fragment of chloroplast DNA (Taberlet. PCR assays The DNA extracts were analyzed by PCR for evaluation of suitability for amplification. Determination of DNA concentration and purity DNA concentration and purity.5 ll of HotStarTaq Master Mix (QIAGEN.6. Moreover. 2. / Food Control 18 (2007) 76–80 2. Pinto et al.5. Amplified products detection PCR amplified products were analysed by electrophoresis on 2. 40 cycles of denaturation at 94 °C for 30 s. Horse meat samples The primer used for horse meat identification. were targeted a 439 bp fragment of mitochondial cytochrome b genes of equine specie. followed by 40 cycles of denaturation at 94 °C for 30 s. 1999). the values of A260 absorbance and A260/280 ratio demonstrated a low DNA extraction efficiency from horse meat and pasteurized milk samples. were discordant. OHIO) and visualized by ethidium bromide staining and UV transilluminator. The homogenate was incubated at 56 °C overnight and then for a further at 70 °C for 1 h with 3 ml Buffer AL. Then the 3 ml mixture was applied to the QIAamp spin column and the DNA was adsorbed onto the QIAamp silica-gel membrane during four centrifugation steps. obtained after centrifugation at 4000g for 5 min at room temperature. except from chocolate cream and maize oil. Hilden. upstream primer 5 0 -gac ctc cca gct cca tca aac atc tca tct tga tga aa-3 0 and downstream primer 5 0 -ctc aga ttc act cga cga ggg tag ta-3 0 (Matsunaga et al. 0. 2. Bovine milk samples The primer pairs used in the study..5 ll of HotStarTaq Master Mix (QIAGEN. the DNA extracts tested negative to spectrophotometer analysis was subjected to PCR analysis using 2–5 ll of template in the reaction mix. 2.3. The A260/280 absorbance ratio was comprised between 0.02 M EDTA.2. Uppsala. The limit of detection (LOD) (lowest order of detection) of different matrices was established by performing 20-fold dilutions from a 1 lg/ll DNA solution. In particular the spectrophotometer analysis on DNA purified by Promega Wizard Magnetic DNA Purification for Food kit gave positive results. Results The comparison of efficiency of the DNA extraction and purification procedures. 1992. Italy) was used for spectrophotometer analysis. 2..5 ll of HotStarTaq Master Mix (QIAGEN. 1 and 2. upstream primers 5 0 -cga aat cgg tag acg cta cg-3 0 and downstream primer 5 0 -ggg gat aga ggg act tga ac-3 0 . annealing at 55 °C for 30 s. Milan.0 (USB.5 lM of each primer and 1 ll of serial dilution of DNA from 1 lg/ll to 6 fg/ll. Hilden.89 M boric acid. 0.5 lM of each primer and 1 ll of serial dilution of DNA from 1 lg/ll to 6 fg/ll. estimated by measuring the A260 absorbance and A260/280 absorbance ratio respectively. & Bouvet.5. The reaction was performed in a final volume of 25 ll using 12. 2. Milan. Cleveland. .

125 pg/ul of DNA. lane 2: 1 ng/ul of DNA. / Food Control 18 (2007) 76–80 79 dumpling. 3. Discussion PCR methods have been successfully applied for the food analysis. lane 9: 2. lane 7: negative control. lane 10: 0. Unlike the WizardÒ Magnetic DNA Purification for Food Promega approach for DNA extraction and purification from lecithin and chocolate.125 pg/ll. PCR assays The PCR assays highlighted that DNA extracts from not processed vegetable samples. lane 12: negative control. vegetable bouillon cube and cherry jam. The PCR detection limits. which allow better DNA binding and removing of inhibitors. the first critical step in molecular analytical methodologies. purification and concentration. such as dumpling. The absorbance value at 260 nm demonstrated that revised DNeasyÒ Tissue Kit was successfully applied to complex and processed matrices. No amplicons were detectable from chocolate. preservatives.5 pg/ul of DNA. The use of a ‘‘mobile solid phase. Moreover. Thus. vegetable bouillon cube and cherry jam. demonstrated more efficiency with complex and processed matrices. lane 6: negative control.05 ng/ll of DNA. were between 0. 4. in this study. lane 4: 2. the DNeasyÒ Tissue Kit (QIAGEN. Germany) system do not involve use Fig. Hilden. chocolate snack and horse meat.8. Discordant data were obtained from spectrophotometer analysis on chocolate cream eluate. lane 8: 0. lane 3: 0. reported in Table 1. Pinto et al. probably due to lower fat content and to higher efficiency in polysaccharides and polyphenolics removing. the Promega Wizard Magnetic DNA Purification for Food approach demonstrated an higher efficiency for vegetable matrices rich in polysaccharides and polyphenolics. leading to ambiguous results. lane 5: 0. lane 2: 1 ng/ll of DNA. such as GMO maize. chocolate snack. lane 7: 1 ng/ll. maize oil. DNA isolation methods for different food sample must provide sufficient removing of food residue. The spectrophotometer analysis gave negative results from maize oil template only. additives. except chocolate cream and maize oil. which might interfere with downstream applications.05 ng/ul of DNA. Lane 1: 100 pb DNA Ladder.D. tested using spiking assay. chocolate snack. provided the best PCR detection limit for vegetable matrices. lane 11: 6 fg/ll.2. This system is less time-consuming and technically demanding than the revised DNeasyÒ Tissue procedure. The A260/ A280 ratio was comprised between 1. 1. were suitable for PCR amplification. Electrophoretic profile of PCR products from maize flour using WizardÒ Magnetic DNA Purification for Food Promega (Lane 2–6) and DNeasyÒ Tissue Kit (Lane 7–11). lane 4: 2.5 pg/ll. lane 3: 0. requires methods able to remove the several inhibitor compounds of the amplification reaction. Electrophoretic profile of PCR products from horse meat. confirming the manufacturing instructions. thanks to their high specificity and sensitivity as well as to their rapidity. was not highlighted for maize oil only.5 pg/ll of DNA.A. The presence of inhibitors in the PCR negative samples. poultry-feed and bran by Promega Wizard Magnetic DNA Purification for Food kit. modified concerning the lysis conditions depending on the size and type of the source material. 1997). In particular. The procedure based on use of the DNeasyÒ Tissue kit. PCR analysis of the samples was negative for the chocolate cream only.125 pg/ll of DNA. 2. the high specificity and the reproducibility of the DNeasyÒ Tissue approach suggest that it is feasible and ideal for routine analysis on dairy and meat products. has highlighted a different efficiency in extraction and removing the inhibitors interfering in the PCR test.5 pg/ll. potato . the PCR assay carried out using 4 ll of maize oil template gave a positive result. The DNA extraction. However.7 and 1. lane 5: 0. The sensitivity. it proved to be simple and reliable for the GMO detection. The evaluation of suitability for mplification performed on DNA extracts obtained using revised DNeasyÒ Tissue Kit gave positive results for all samples. maize flour. Lane 1: 100 pb DNA Ladder. food processing may degrade DNA molecules and introduce substances that interfere with the PCR reaction.’’ unlike columnbased system. such as dumpling. Fig.05 ng/ll and 2. lane 6: 6 fg/ll of DNA.05 ng/ll. but it is mainly limited by the presence of inhibitors (Wilson. probably due to the buffering conditions and silica-column-based system. The comparison of two extraction methods.

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