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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
` 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: firstname.lastname@example.org (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
1 9.3–37..05 ng/ll 2. Then.37–1.2 9.. 2.0 0 328.05 ng/ll ND 2.1–0.8–467.67 1.9–80.78 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.37 1.3.75–1.1. The tube was mixed and replaced on MagneSphereÒ Magnetic Separation Stand (Promega Italia S. After 1 min incubation at room temperature. Positive controls DNA from Certiﬁed Reference Material IRMM-411R-4 2% Bt-176 maize (Fluka. Samples Food and feed samples listed in Table 1 were used in this study. Hilden.11 1.0 62.2–44.r.2 40. Then 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.88–1.5 14.2 28.2 37.5 pg ng/ll 0.3 139. The tube was mixed and incubated at room temperature for 5 min by shaking.7 2–4.06 0 0 0.08 0. Madrid. The supernatant was added to 50 ll of resuspended MagneSilTM PMPs and 1 ml of isopropanol was added. 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.2–64.6 13. Italy).8–93.125 pg/ll ND 0.3 48.1 38.70–1.1 50.1.62–1.8 102.5–128. 100 ll of nuclease-free water was added to particles and the mixture obtained was mixed and incubated at 65 °C for 5 min.5 125. Milano.8 3. Milano. Germany).0–1. Pinto et al.2 59. from horse and cow (BIOTOOLS B&M Labs.3–56.5 67.95–1.5 pg/ll 0.125 pg/ll 2.. after 1 min in the magnetic stand.89–1.39 0.1 1.7–14.D. 2.7 12. The tube was placed onto the MagneSphereÒ Magnetic Separation Stand (Promega Italia S. the liquid phase was discarded.8 15.2 0.3 0.4 106.6 100. The mixture obtained was centrifuged for 10 min at 13.r.7 1.56–2.59–1. Milano.3 0.2 38. Italy) and DNeasyÒ Tissue Kit (QIAGEN.3–52.8 48. 2.0 1..000g.05 ng/ll ND 2.00 1. Table 1 Results Sample (200 mg) Extraction methods DNA concentration range (ng/ll) Puriﬁcation Puriﬁcation Puriﬁcation Puriﬁcation Puriﬁcation 98.5 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 .11–1.25–2.A.l.05–2.77 DNA concentration media 60.l.6–19.0–8. incubated for 10 min at room temperature with 250 ll of Buﬀer B.05 ng/ll ND ND ND Amplicon detected with 4 ll of template 0. Material and methods 2.125 pg/ll 2. was added to 750 ll of precipitation solution. The liquid phase was discarded leaving the tubes in the stand. Then sample. The ﬁnal volume was adjusted to 100 ll by adding nuclease-free water.04 0 1.l.8–20.6 8.5 11.43 1.4 0 0 26.40–1.97 0.8–70 96.5 pg/ll 2. This step was repeated for three times and in the end the particles were dried at room temperature for 15–30 min.5 pg/ll 0. Geel.4 46.76–1.13–1.0 0–0.5 11.7–38.46–1.2 28.3 0 376.2.4–69. Belgium).6 98. 2. Spain) were used as positive controls.5 pg/ll 1 ng/ll 0.0–50 0–12. Italy) and left in place for 1 min.4 90. / Food Control 18 (2007) 76–80 77 of extraction and puriﬁcation procedures was performed from food and feed samples.3.5 pg/ll 1 ng/ll 2.99–1.2 8–10 A260/280 range 1.r.l.4 1. the liquid phase was discarded.9 3.85–2.5 26. The tube was removed from the stand and 250 ll of lysis buﬀer B was added to the particles.47 1. 1 ml of 70% ethanol wash solution was added and.05 ng/ll 0.r.2–141. Milano.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 0.6 0 0 20.
the values of A260 absorbance and A260/280 ratio demonstrated a low DNA extraction eﬃciency from horse meat and pasteurized milk samples. were targeted a fragment of chloroplast DNA (Taberlet. were: denaturation step of 95 °C for 15 min.89 M Tris. Results The comparison of eﬃciency of the DNA extraction and puriﬁcation procedures. Hilden.97. 2. gave the results reported in Table 1 and in Figs.1. except from chocolate cream and maize oil. 2. Vegetable samples PCR test of vegetable samples was carried out using primer pair. The PCR reactions which gave no amplicons were subjected to spiked controls for identiﬁcation the presence of PCR inhibitors. Horse meat samples The primer used for horse meat identiﬁcation. The reaction was performed in a ﬁnal volume of 25 ll using 12. the volume of Proteinase K and lysis buﬀers and the number of washing steps. 0.5 lM of each primer and 1 ll of serial dilution of DNA from 1 lg/ll to 6 fg/ll. extension at 72 °C of 30 s and a ﬁnal extension at 72 °C for 7 min. Vilnius.. pH 8.5. A spectrophotometer BioPhotometer (Eppendorf. Moreover.1 and 1. 40 cycles of denaturation at 94 °C for 30 s. DNA extraction by DNeasyÒ Tissue Kit The protocol for puriﬁcation of total DNA from animal tissue was revised concerning the quantiﬁcation of starting material. In particular the spectrophotometer analysis on DNA puriﬁed by Promega Wizard Magnetic DNA Puriﬁcation for Food kit gave positive results. 2. The puriﬁed DNA was eluted from the QIAamp spin column in 100 ll of elution buﬀer. The reaction was performed in a ﬁnal volume of 25 ll using 12.5.5 lM of each primer and 1 ll of serial dilution of DNA from 1 lg/ll to 6 fg/ll.5 lM of each primer and 1 ll of serial dilution of DNA from 1 lg/ll to 6 fg/ll.4. were targeted a 279 bp fragment of mitochondrial cytochrome b gene of bovine specie. 2002).5 ll of HotStarTaq Master Mix (QIAGEN. The reaction was performed in a ﬁnal volume of 25 ll using 12. Cleveland. Pinto et al. annealing at 55 °C for 30 s. Germany).3. OHIO) and visualized by ethidium bromide staining and UV transilluminator. Then DNA bound to the QIAamp membrane was washed by centrifugation using two diﬀerent washing buﬀers. Lithuania) was used as marker. 1 and 2.5. PCR assays The DNA extracts were analyzed by PCR for evaluation of suitability for ampliﬁcation. The cycling conditions for the PCR assays.5 ll of HotStarTaq Master Mix (QIAGEN. Sweden) gel in 1· TBE buﬀer containing 0. Italy) with an initial denaturation step of 95 °C for 15 min.1. Germany). 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.. obtained after centrifugation at 4000g for 5 min at room temperature. 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.78 A. Determination of DNA concentration and purity DNA concentration was determined by measuring the absorbance at 260 nm. Uppsala. . followed by 40 cycles of denaturation at 94 °C for 30 s.). A Gene RulerTM 100 bp DNA Ladder Plus (MBI Fermentas. were targeted a 439 bp fragment of mitochondial cytochrome b genes of equine specie. The PCR was processed in a Mastercycler 5332 (Eppendorf. Italy) was used for spectrophotometer analysis. was added to 3 ml ethanol. 0. 3. 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. Milan. 200 mg of sample were homogenized with 3 ml of lysis buﬀer ATL and 100 ll proteinase K (20 mg/ml). The supernatant. 1992. 1999).2. Italy). The A260/280 absorbance ratio was comprised between 0.0 (USB. were discordant. carried out by measuring the DNA concentration and suitability for PCR. 2. Hilden. 0. 0.89 M boric acid. Bovine milk samples The primer pairs used in the study. estimated by measuring the A260 absorbance and A260/280 absorbance ratio respectively.5% (w/v) agarose NA (Pharmacia. In particular. Ampliﬁed products detection PCR ampliﬁed products were analysed by electrophoresis on 2. 3. 2. Hilden. DNA purity was measured by calculating the ratio of absorbance at 260–280 nm. the DNA extracts tested negative to spectrophotometer analysis was subjected to PCR analysis using 2–5 ll of template in the reaction mix. Milan. 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. Milan. Gielly. 2.3.D. Germany).02 M EDTA.2. / Food Control 18 (2007) 76–80 2. Determination of DNA concentration and purity DNA concentration and purity. & Bouvet. 0.5. Moreover. carried out in a Mastercycler 5332 (Eppendorf.5 ll of HotStarTaq Master Mix (QIAGEN. 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. The cycling conditions for the PCR assay were the same of the program used for Bovine milk samples. 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 .6.
Moreover. but it is mainly limited by the presence of inhibitors (Wilson. were suitable for PCR ampliﬁcation. lane 3: 0.125 pg/ul of DNA. Germany) system do not involve use Fig.125 pg/ll.5 pg/ul of DNA.7 and 1.5 pg/ll of DNA.’’ unlike columnbased system. lane 2: 1 ng/ll of DNA. thanks to their high speciﬁcity and sensitivity as well as to their rapidity. / Food Control 18 (2007) 76–80 79 dumpling. requires methods able to remove the several inhibitor compounds of the ampliﬁcation reaction. lane 5: 0. 4. chocolate snack and horse meat. tested using spiking assay. lane 3: 0. lane 9: 2. The sensitivity. Pinto et al. 1997). such as dumpling.125 pg/ll of DNA.A. food processing may degrade DNA molecules and introduce substances that interfere with the PCR reaction.D. such as dumpling. vegetable bouillon cube and cherry jam. The comparison of two extraction methods. lane 6: 6 fg/ll of DNA. modiﬁed concerning the lysis conditions depending on the size and type of the source material. lane 4: 2. In particular. was not highlighted for maize oil only. Lane 1: 100 pb DNA Ladder. the PCR assay carried out using 4 ll of maize oil template gave a positive result.05 ng/ll and 2. However. has highlighted a diﬀerent eﬃciency in extraction and removing the inhibitors interfering in the PCR test.05 ng/ll. lane 2: 1 ng/ul of DNA. preservatives. the DNeasyÒ Tissue Kit (QIAGEN. The spectrophotometer analysis gave negative results from maize oil template only. which allow better DNA binding and removing of inhibitors.5 pg/ll. in this study.8. lane 12: negative control. probably due to lower fat content and to higher eﬃciency in polysaccharides and polyphenolics removing. The use of a ‘‘mobile solid phase. The presence of inhibitors in the PCR negative samples. lane 11: 6 fg/ll. lane 5: 0. The A260/ A280 ratio was comprised between 1. probably due to the buﬀering conditions and silica-column-based system. additives. except chocolate cream and maize oil. Hilden. lane 8: 0. 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. Fig. potato . The absorbance value at 260 nm demonstrated that revised DNeasyÒ Tissue Kit was successfully applied to complex and processed matrices. lane 7: negative control. The procedure based on use of the DNeasyÒ Tissue kit. The DNA extraction. 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). chocolate snack. This system is less time-consuming and technically demanding than the revised DNeasyÒ Tissue procedure. Unlike the WizardÒ Magnetic DNA Puriﬁcation for Food Promega approach for DNA extraction and puriﬁcation from lecithin and chocolate. Thus. the ﬁrst critical step in molecular analytical methodologies. The evaluation of suitability for mpliﬁcation performed on DNA extracts obtained using revised DNeasyÒ Tissue Kit gave positive results for all samples. 3. Discussion PCR methods have been successfully applied for the food analysis. the Promega Wizard Magnetic DNA Puriﬁcation for Food approach demonstrated an higher eﬃciency for vegetable matrices rich in polysaccharides and polyphenolics. chocolate snack. PCR assays The PCR assays highlighted that DNA extracts from not processed vegetable samples. lane 10: 0. puriﬁcation and concentration. maize oil. lane 7: 1 ng/ll. such as GMO maize. vegetable bouillon cube and cherry jam. which might interfere with downstream applications.5 pg/ll. demonstrated more eﬃciency with complex and processed matrices. 2.05 ng/ul of DNA.2. PCR analysis of the samples was negative for the chocolate cream only. 1. Electrophoretic proﬁle of PCR products from horse meat.05 ng/ll of DNA. lane 4: 2. The PCR detection limits. provided the best PCR detection limit for vegetable matrices. Lane 1: 100 pb DNA Ladder. poultry-feed and bran by Promega Wizard Magnetic DNA Puriﬁcation for Food kit. No amplicons were detectable from chocolate. conﬁrming the manufacturing instructions. maize ﬂour. it proved to be simple and reliable for the GMO detection. Discordant data were obtained from spectrophotometer analysis on chocolate cream eluate. leading to ambiguous results. DNA isolation methods for different food sample must provide suﬃcient removing of food residue. lane 6: negative control. were between 0. reported in Table 1.
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