Food Control 18 (2007) 7680 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 purication procedures for food analysis, Wizard Magnetic DNA Purication for Food (Promega) and DNeasy Tissue Kit (QIAGEN), have been compared concerning extraction eciency, DNA purity and DNA suitability for amplication. The comparison of two extraction methods, in this study, has highlighted the eciency of the Wizard Magnetic DNA Purication approach for vegetable matrices while the revised DNeasy Tissue Kit for complex and processed matrices. 2005 Elsevier Ltd. All rights reserved.
Keywords: Food; DNA extraction; DNA purication; 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 specic, 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 nal 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 purication techniques. In particular, the application of molecular methods to food samples requires stringent extraction and purication strategies that ensure ecient 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 purication of nucleic acids from dierent 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 purication procedures for food analysis, Wizard Magnetic DNA Purication for Food (Promega Italia S.r.l., Milano, Italy) and DNeasy Tissue Kit (QIAGEN, Hilden, Germany), have been compared concerning extraction eciency, DNA purity and DNA suitability for amplication. Although both approaches involve the extraction of DNA using the silica as anity matrix, one procedure uses a mobile solid phase, while the other is a column-based system. The comparison

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of extraction and purication procedures was performed from food and feed samples. 2. Material and methods 2.1. Samples Food and feed samples listed in Table 1 were used in this study. 2.2. Positive controls DNA from Certied Reference Material IRMM-411R-4 2% Bt-176 maize (Fluka, Geel, Belgium), from horse and cow (BIOTOOLS B&M Labs, Madrid, Spain) were used as positive controls. 2.3. DNA extraction DNA was extracted and puried using two commercial kits based on use of silica as anity matrix: Wizard Magnetic DNA Purication for Food (Promega Italia S.r.l., Milano, Italy) and DNeasy Tissue Kit (QIAGEN, Hilden, Germany). 2.3.1. DNA extraction by Wizard magnetic DNA purication for food Two hundred milligram of sample were vortexed vigorously with 500 ll of lysis buer A and 5 ll of RNase A.

Then sample, incubated for 10 min at room temperature with 250 ll of Buer B, was added to 750 ll of precipitation solution. The mixture obtained was centrifuged for 10 min at 13.000g. The supernatant was added to 50 ll of resuspended MagneSilTM PMPs and 1 ml of isopropanol was added. The tube was mixed and incubated at room temperature for 5 min by shaking. Then the tube was placed onto the MagneSphere Magnetic Separation Stand (Promega Italia S.r.l., Milano, Italy) and left in place for 1 min. The liquid phase was discarded leaving the tubes in the stand. The tube was removed from the stand and 250 ll of lysis buer B was added to the particles. The tube was mixed and replaced on MagneSphere Magnetic Separation Stand (Promega Italia S.r.l., Milano, Italy). After 1 min incubation at room temperature, the liquid phase was discarded. Then, 1 ml of 70% ethanol wash solution was added and, after 1 min in the magnetic stand, the liquid phase was discarded. This step was repeated for three times and in the end the particles were dried at room temperature for 1530 min. 100 ll of nuclease-free water was added to particles and the mixture obtained was mixed and incubated at 65 C for 5 min. The tube was placed onto the MagneSphere Magnetic Separation Stand (Promega Italia S.r.l., Milano, 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. The nal volume was adjusted to 100 ll by adding nuclease-free water.

Table 1 Results Sample (200 mg) Extraction methods DNA concentration range (ng/ll) Purication Purication Purication Purication Purication 98.352.4 46.738.2 37.264.7 12.619.3 139.893.4 106.980.0 0 328.8467.4 0 0 26.714.5 11.08.1 38.5128.6 8.820.0 62.337.5 26.870 96.2141.5 11.244.5 125.469.6 13.050 012.5 67.356.7 24.2 810 A260/280 range 1.461.11 1.052.1 1.111.97 0.881.47 1.891.37 1.252.04 0 1.131.06 0 0 0.371.08 0.951.2 0.401.0 1.01.39 0.701.3 0.991.3 0.591.4 1.852.00 1.621.43 1.562.0 00.7 1.761.78 0.10.67 1.751.77 DNA concentration media 60.2 40.3 48.5 14.8 102.4 90.3 0 376.6 0 0 20.2 9.1 50.8 15.8 48.2 38.6 100.2 28.6 98.2 28.9 3.2 59.8 3.1 9.1 PCR LOD Spike test

IRMM-411R-4 2% Bt-176 Maize our Poultry feed Chocolate cream Mays oil

Wizard Magnetic DNA DNeasy Tissue kit Wizard Magnetic DNA DNeasy Tissue kit Wizard Magnetic DNA DNeasy Tissue kit Wizard Magnetic DNA DNeasy Tissue kit Wizard Magnetic DNA DNeasy Tissue kit Wizard Magnetic DNA DNeasy Tissue kit Wizard Magnetic DNA DNeasy Tissue kit Wizard Magnetic DNA DNeasy Tissue kit Wizard Magnetic DNA DNeasy Tissue kit Wizard Magnetic DNA DNeasy Tissue kit Wizard Magnetic DNA DNeasy Tissue kit Wizard Magnetic DNA DNeasy Tissue kit

Cherry marmalade Potato dumpling Chocolate snack Vegetable bouillon cube Bran Horse meat Pasteurised milk

Purication Purication Purication Purication Purication Purication Purication

0.125 pg/ll 2.5 pg/ll 0.125 pg/ll 2.5 pg/ll 2.5 pg ng/ll 0.05 ng/ll ND ND ND Amplicon detected with 4 ll of template 0.05 ng/ll 0.125 pg/ll ND 0.05 ng/ll ND 2.5 pg/ll 1 ng/ll 0.05 ng/ll 2.5 pg/ll 0.05 ng/ll ND 2.5 pg/ll 1 ng/ll 2.5 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

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2.3.2. DNA extraction by DNeasy Tissue Kit The protocol for purication of total DNA from animal tissue was revised concerning the quantication of starting material, the volume of Proteinase K and lysis buers and the number of washing steps. In particular, 200 mg of sample were homogenized with 3 ml of lysis buer ATL and 100 ll proteinase K (20 mg/ml). The homogenate was incubated at 56 C overnight and then for a further at 70 C for 1 h with 3 ml Buer AL. The supernatant, obtained after centrifugation at 4000g for 5 min at room temperature, was added to 3 ml ethanol. 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. Then DNA bound to the QIAamp membrane was washed by centrifugation using two dierent washing buers. The puried DNA was eluted from the QIAamp spin column in 100 ll of elution buer. 2.4. Determination of DNA concentration and purity DNA concentration was determined by measuring the absorbance at 260 nm. DNA purity was measured by calculating the ratio of absorbance at 260280 nm. A spectrophotometer BioPhotometer (Eppendorf, Milan, Italy) was used for spectrophotometer analysis. 2.5. PCR assays The DNA extracts were analyzed by PCR for evaluation of suitability for amplication. The limit of detection (LOD) (lowest order of detection) of dierent matrices was established by performing 20-fold dilutions from a 1 lg/ll DNA solution. Moreover, the DNA extracts tested negative to spectrophotometer analysis was subjected to PCR analysis using 25 ll of template in the reaction mix. The PCR reactions which gave no amplicons were subjected to spiked controls for identication the presence of PCR inhibitors. 2.5.1. Vegetable samples PCR test of vegetable samples was carried out using primer pair, 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 , were targeted a fragment of chloroplast DNA (Taberlet, Gielly, & Bouvet, 1992.). The reaction was performed in a nal volume of 25 ll using 12.5 ll of HotStarTaq Master Mix (QIAGEN, Hilden, Germany), 0.5 lM of each primer and 1 ll of serial dilution of DNA from 1 lg/ll to 6 fg/ll. The cycling conditions for the PCR assays, carried out in a Mastercycler 5332 (Eppendorf, Milan, Italy), were: denaturation step of 95 C for 15 min, 40 cycles of denaturation at 94 C for 30 s, annealing at 55 C for 30 s, extension at 72 C of 30 s and a nal extension at 72 C for 7 min. 2.5.2. Bovine milk samples The primer pairs used in the study, 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., 2002), were targeted a 279 bp fragment of mitochondrial cytochrome b gene of bovine specie. The reaction was performed in a nal volume of 25 ll using 12.5 ll of HotStarTaq Master Mix (QIAGEN, Hilden, Germany), 0.5 lM of each primer and 1 ll of serial dilution of DNA from 1 lg/ll to 6 fg/ll. The PCR was processed in a Mastercycler 5332 (Eppendorf, Milan, Italy) with an initial denaturation step of 95 C for 15 min, followed by 40 cycles of denaturation at 94 C for 30 s, annealing at 58 C for 30 s and extension at 72 C for 30 s and a nal extension at 72 C for 7 min. 2.5.3. Horse meat samples The primer used for horse meat identication, 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., 1999), were targeted a 439 bp fragment of mitochondial cytochrome b genes of equine specie. The reaction was performed in a nal volume of 25 ll using 12.5 ll of HotStarTaq Master Mix (QIAGEN, Hilden, Germany), 0.5 lM of each primer and 1 ll of serial dilution of DNA from 1 lg/ll to 6 fg/ll. The cycling conditions for the PCR assay were the same of the program used for Bovine milk samples. 2.6. Amplied products detection PCR amplied products were analysed by electrophoresis on 2.5% (w/v) agarose NA (Pharmacia, Uppsala, Sweden) gel in 1 TBE buer containing 0.89 M Tris, 0.89 M boric acid, 0.02 M EDTA, pH 8.0 (USB, Cleveland, OHIO) and visualized by ethidium bromide staining and UV transilluminator. A Gene RulerTM 100 bp DNA Ladder Plus (MBI Fermentas, Vilnius, Lithuania) was used as marker. 3. Results The comparison of eciency of the DNA extraction and purication procedures, carried out by measuring the DNA concentration and suitability for PCR, gave the results reported in Table 1 and in Figs. 1 and 2. 3.1. Determination of DNA concentration and purity DNA concentration and purity, estimated by measuring the A260 absorbance and A260/280 absorbance ratio respectively, were discordant. In particular the spectrophotometer analysis on DNA puried by Promega Wizard Magnetic DNA Purication for Food kit gave positive results, except from chocolate cream and maize oil. The A260/280 absorbance ratio was comprised between 0.1 and 1.97. Moreover, the values of A260 absorbance and A260/280 ratio demonstrated a low DNA extraction eciency from horse meat and pasteurized milk samples.

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dumpling, chocolate snack and horse meat. The presence of inhibitors in the PCR negative samples, tested using spiking assay, was not highlighted for maize oil only. The evaluation of suitability for mplication performed on DNA extracts obtained using revised DNeasy Tissue Kit gave positive results for all samples, except chocolate cream and maize oil. However, the PCR assay carried out using 4 ll of maize oil template gave a positive result. PCR analysis of the samples was negative for the chocolate cream only. 4. Discussion PCR methods have been successfully applied for the food analysis, thanks to their high specicity and sensitivity as well as to their rapidity, but it is mainly limited by the presence of inhibitors (Wilson, 1997). Moreover, food processing may degrade DNA molecules and introduce substances that interfere with the PCR reaction, leading to ambiguous results. Thus, DNA isolation methods for different food sample must provide sucient removing of food residue, additives, preservatives, which might interfere with downstream applications. The DNA extraction, purication and concentration, the rst critical step in molecular analytical methodologies, requires methods able to remove the several inhibitor compounds of the amplication reaction. The comparison of two extraction methods, in this study, has highlighted a dierent eciency in extraction and removing the inhibitors interfering in the PCR test. In particular, the Promega Wizard Magnetic DNA Purication for Food approach demonstrated an higher eciency for vegetable matrices rich in polysaccharides and polyphenolics. This system is less time-consuming and technically demanding than the revised DNeasy Tissue procedure; it proved to be simple and reliable for the GMO detection, conrming the manufacturing instructions. The use of a mobile solid phase, unlike columnbased system, provided the best PCR detection limit for vegetable matrices, probably due to lower fat content and to higher eciency in polysaccharides and polyphenolics removing. The procedure based on use of the DNeasy Tissue kit, modied concerning the lysis conditions depending on the size and type of the source material, demonstrated more eciency with complex and processed matrices, such as dumpling, chocolate snack, vegetable bouillon cube and cherry jam, probably due to the buering conditions and silica-column-based system, which allow better DNA binding and removing of inhibitors. The sensitivity, the high specicity and the reproducibility of the DNeasy Tissue approach suggest that it is feasible and ideal for routine analysis on dairy and meat products. Unlike the Wizard Magnetic DNA Purication for Food Promega approach for DNA extraction and purication from lecithin and chocolate, the DNeasy Tissue Kit (QIAGEN, Hilden, Germany) system do not involve use

Fig. 1. Electrophoretic prole of PCR products from horse meat. Lane 1: 100 pb DNA Ladder; lane 2: 1 ng/ul of DNA; lane 3: 0.05 ng/ul of DNA; lane 4: 2.5 pg/ul of DNA; lane 5: 0.125 pg/ul of DNA; lane 6: negative control; lane 7: negative control.

Fig. 2. Electrophoretic prole of PCR products from maize our using Wizard Magnetic DNA Purication for Food Promega (Lane 26) and DNeasy Tissue Kit (Lane 711). Lane 1: 100 pb DNA Ladder; lane 2: 1 ng/ll of DNA; lane 3: 0.05 ng/ll of DNA; lane 4: 2.5 pg/ll of DNA; lane 5: 0.125 pg/ll of DNA; lane 6: 6 fg/ll of DNA; lane 7: 1 ng/ll; lane 8: 0.05 ng/ll; lane 9: 2.5 pg/ll; lane 10: 0.125 pg/ll; lane 11: 6 fg/ll; lane 12: negative control.

The absorbance value at 260 nm demonstrated that revised DNeasy Tissue Kit was successfully applied to complex and processed matrices, such as dumpling, chocolate snack, vegetable bouillon cube and cherry jam. The A260/ A280 ratio was comprised between 1.7 and 1.8. The spectrophotometer analysis gave negative results from maize oil template only. Discordant data were obtained from spectrophotometer analysis on chocolate cream eluate. 3.2. PCR assays The PCR assays highlighted that DNA extracts from not processed vegetable samples, such as GMO maize, maize our, poultry-feed and bran by Promega Wizard Magnetic DNA Purication for Food kit, were suitable for PCR amplication. The PCR detection limits, reported in Table 1, were between 0.05 ng/ll and 2.5 pg/ll. No amplicons were detectable from chocolate, maize oil, potato

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A.D. Pinto et al. / Food Control 18 (2007) 7680 Lewis, G. D., & Metcalf, T. G. (1988). Polyethylene glycol precipitation for recovery of pathogenic viruses, including hepatitis A virus and human rotavirus, from oyster, water and sediment samples. Applied and Environment Microbiology, 54, 19831988. Lipp, M., Bluth, A., Eyquem, F., Kruse, L., Schimmel, H., Van den Eede, G., et al. (2001). Validation of a method based on polymerase chain reaction for the detection of genetically modied organism in various processed foodstu. European Food Research Technology, 212, 497504. Lockley, A. K., & Bardsley, R. G. (2000). DNA-based methods for food authentication. Trend Food Science, XI, 6777. Matsunaga, T., Chikuni, K., Tanabe, R., Muroya, S., Shibata, K., Yamada, J., et al. (1999). A quick and simple method for the identication of meat species and meat products by PCR assay. Meat Science, 511, 43148. Meyer, R. (1999). Development and application of DNA analytical methods for the detection of GMOs in food. Food Control, 10, 391399. Meyer, R., & Candrian, U. (1996). PCR-based DNA analysis for the identication and characterization of food components. Lebensmittel Wissenschaft Technologie, 29, 19. Pan, T. M. (2002). Current status and detection of genetically modied organism. Journal of Food and Drug Analysis, 10, 229241. Schwab, K. J., Neill, F. H., Le Guyader, F., Estes, M. K., & Atmar, R. L. (2001). Development of a reverse transcription-PCR-DNA enzyme immunoassay for detection of Norwalk-like viruses and hepatitis A virus in stool and shellsh. Applied and Environment Microbiology, 67, 742749. Soule, H., Genoulaz, O., Gratacap-Cavallier, B., Chevallier, P., Liu, J. X., & Seigneurin, J. M. (2000). Ultraltration and reverse transcriptionpolymerase chain reaction: An ecient process for poliovirus, rotavirus and hepatitis a virus detection in water. Water Research, 34, 10631067. Taberlet, P., Gielly, L., & Bouvet, J. (1992). Universal primers for amplication of three non-coding regions of chloroplast DNA. Plant Molecular Biology, 17, 11051109. Wilson, I. G. (1997). Inhibition and facilitation of nucleic acid amplication. Applied and Environment Microbiology, 63, 37413751. Zimmermann, A., Luthy, J., & Pauli, U. (1998). Quantitative and qualitative evaluation of nine dierent methods for nucleic acids on soya bean food samples. Zeitschrift fur Lebensmittel-Untersuchung und Forschung A, 207, 8190.

of organic solvents such as hexane, which may be inhibitory to downstream PCR applications. Although the results obtained are quite variable and often extremely low, the DNeasy Tissue adapted protocol showed a higher anity for animal tissues and complex matrices. The study highlighted the need of suitable extraction methods to obtain highly puried nucleic acids without inhibitors, which if not removed may be inhibitory to downstream PCR applications. The Wizard Magnetic DNA Purication for Food Promega approach and the DNeasy Tissue Kit (QIAGEN, Hilden, Germany) gave satisfactory results for most food samples, with successful purication and a lower amount of potential contaminants compared to other techniques, such as the CTAB-based extractions, which may have contaminants of CTAB that can lead to a reducing of the eciency of downstream reactions (data not shown). Further studies are required to build specic PCR-based methods, which could oer a valid approach for guaranteeing an ecient food and feed surveillance system. References
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