Food Chemistry 145 (2014) 1072–1075

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Food Chemistry

journal homepage: www.elsevier .com/locate / foodchem

Analytical Methods

Detection of genetically modified soybean in crude soybean oil

0308-8146/$ - see front matter � 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.foodchem.2013.09.017

⇑ Corresponding author. Tel.: +381 21 4898 150; fax: +381 21 421 249.E-mail address: zorica.nikolic@ifvcns.ns.ac.rs (Z. Nikolic).

Zorica Nikolic a,⇑, Ivana Vasiljevic b, Gordana Zdjelar a, Vuk Ðor -devic a, Maja Ignjatov a, Dušica Jovicic a,Dragana Miloševic a

a Institute of Field and Vegetable Crops, Maksima Gorkog 30, Novi Sad, Serbiab A Bio Tech Lab, Vojvode Putnika bb, Sremska Kamenica, Serbia

a r t i c l e i n f o

Article history:Received 30 April 2012Received in revised form 29 August 2013Accepted 4 September 2013Available online 11 September 2013

Keywords:GMOSoybeanCrude oil

a b s t r a c t

In order to detect presence and quantity of Roundup Ready (RR) soybean in crude oil extracted fromsoybean seed with a different percentage of GMO seed two extraction methods were used, CTAB andDNeasy Plant Mini Kit. The amplifications of lectin gene, used to check the presence of soybean DNA,were not achieved in all CTAB extracts of DNA, while commercial kit gave satisfactory results. Comparingactual and estimated GMO content between two extraction methods, root mean square deviation for kitis 0.208 and for CTAB is 2.127, clearly demonstrated superiority of kit over CTAB extraction. The results ofquantification evidently showed that if the oil samples originate from soybean seed with varying percent-age of RR, it is possible to monitor the GMO content at the first stage of processing crude oil.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

The commercial cultivation of genetically modified (GMO) soy-bean varieties began in 1996, and they became predominant in themajor soybean producing countries. Today, around 148 million haof GMO plants are grown and traded, among which about 71% issoybean (James, 2011). The Roundup Ready soybean (RRS), eventGTS 40-3-2, is mainly produced by USA, Argentina and Brazil andexporting in EU and other countries, mostly as soybean meal andcrude vegetable oils, among other products.

Soybean represents about 60% of total oil consumption andusage of GMO soybean seeds for soybean oil production has beencontinuously increasing. Soybean oil is the most highly consumedvegetable oil worldwide. It appears in a wide variety of processedfoods and in industrial products such as fatty acids, soaps andbiodiesel.

The European Union (EU) has established the legal basis for thetraceability and labelling requirements of genetically modifiedorganisms and GMO derived food and feeds (Regulations (EC) No.1829/2003, 1830/2003). Furthermore, it covers products destinedfor industrial processing for uses other than consumption (e.g. inthe production of a biofuel). Traceability implies a system to docu-ment the history of product of the direction from primary rawmaterials to the finishing consumable (MacDaniel & Sheridan,2001). In this sense, traceability is needed for all products, whichare tradable.

For labelling of highly processed products in which the GMOprotein or DNA may be undetectable, to trace the GMO status ofthe product traceability is especially useful (Davidson & Bertheau,2007).

There are few reports concerning extraction of DNA and detec-tion of GMO in highly processed food such as oil. The initial reportpresented that no genetic material can be recovered after the firstprocessing steps of soybean oil, and negative result for all fractionsof industrially produced soybean oil (Pauli, Liniger, & Zimmer-mann, 1998). In opposition to this successful detection of DNAfragments in samples of cold pressed oil, as well as in samples ofrefined oil, have been reported by Hellebrand, Nagy, and Mörsel(1998). Refining process has influence upon the quality andquantity of DNA, and after the degumming DNA was concentratedin water fraction, no DNA could be amplified in the oil fraction(Gryson et al., 2002).

A more recent study comparing four different extraction proto-cols to recover DNA in soybean oil showed that the choice of theextraction method was a critical parameter to detect a specificDNA fragment by PCR and real-time PCR (Costa, Mafra, Amaral,Beatriz, & Oliveira, 2010a). The other study demonstrated that itis possible to extract trace amounts of amplifiable DNA along acomplete industrial soybean oil processing chain mostly based onthe use of the commercial kit for DNA extraction (Costa, Mafra,Amaral, & Oliveira, 2010b). DNA could be recovered from distincttypes of vegetable oil (soybean, rapeseed, maize and flax) at differ-ent stages of the oil refining, even in refined oil at least for highcopy DNA targets (Debode, Janssen, & Berbe, 2012).

Mentioned authors have been working with oil samplesobtained from soybean seed with a high percentage, around 50%

Z. Nikolic et al. / Food Chemistry 145 (2014) 1072–1075 1073

or 80% of Roundup Ready soybean. There is no report if it is possi-ble to detect and quantify the GMO if the oil samples wereobtained from seed, which contain smaller amount of RRS seed.This could be a very practical concern because in seed production,transport and storage, some mixtures of non GMO and GM soybeanare likely to occur (Bullock & Desquilbet, 2002). The aim of thisstudy was to detect and estimate the quantity of Roundup Readysoybean in crude oil extracted from soybean seed with various per-centages of GMO seed using two extraction methods.

2. Materials and methods

2.1. Samples

Soybean seed of non GMO variety (Vojvo -danka, Institute ofField and Vegetable Crops, Novi Sad) and GMO variety, modifica-tion GTS 40-3-2 (Roundup Ready, Monsanto), were used in orderto make samples with a different percentage of GMO: 0%, 1%, 5%,10% and 100%. Seed was ground in Thermomix TM21, Vorwerk(Germany). In the preparation of each level, appropriate amountsof the ground sample of GMO soybean and non-GMO soybean wereweighed and mixed thoroughly. Samples were made in tworeplicates.

The Certified reference materials (CRM) were used as driedsoybean powder (GTS 40-3-2) with 0.1% and 0% GMO soybean,developed by the Institute for Reference Materials and Measure-ments (IRMM, Belgium). Unlabeled crude soybean oil bought froma local market was used as additional negative control.

2.2. DNA extraction from oil

100 g of each sample was mixed with n-hexane in ratio 1:10 (v/w), and vortex about 2 h. Solution was filtrated and filtrate wasstirred overnight at room temperature in order to evaporated n-hexane. About 14 ml of crude soybean oil was separated into twotubes and centrifuged at 11,000g for 30 min at 4 �C. After centrifu-gation, the supernatant was discarded. The DNA was extractedfrom a pellet on two ways, using the cetyltrimethylammoniumbromide (CTAB) method (Querci, Jermini, & Van den Eede, 2004)and DNeasy Plant Mini Kit (Qiagen GmbH). The extractions weredone in duplicate assays for each sample.

The quality and purity of DNA were analysed by spectropho-tometry using BioSpec-nano, Shimadzu (Shimadzu Coorporation,Japan). DNA concentrations were determined by UV absorbanceat 260 nm. The purity of the DNA was determined by a ratio ofthe absorbance at 260, 280 and 230 nm. All samples were dilutedwith nuclease-free water up to 50 ng/ll.

2.3. Qualitative PCR

The sequences of oligonucleotide primers used in this work arepresented in Table 1. The primers were synthesized by MetabionInternational AG (Martinsried, Germany).

Table 1Oligonucleotide primers.

Primer Sequence (50–30) Fragment leng

GM03 GCC CTC TAC TCC ACC CCC ATC 118GM04 GCC CAT CTG CAA GCC TTT TTG TG

Le1 GAC GCT ATT GTG ACC TCC TC 318Le6 GAA AGT GTC AAG CTT AAC AGC GACG

35s-f2 TGA TGT GAT ATC TCC ACT GAC G 172petu-r1 TGT ATC CCT TGA GCC ATG TTG T

The PCR was carried out using premix of 2� PCR Master Mix,(Fermentas, Lithuania) containing 4 mM MgCl, 0.4 mM dNTP,0.05 units/ll Taq DNA Polymerase (recombinant).

PCR was performed in a final volume of 25 ll of PCR mix con-taining 0.2 pmol/ll primers for lectin gene and RR soybean and ap-prox. first 100 ng and then 50 ng DNA was used.

Amplifications were carried out in a Mastercycler ep gradient Stermocycler (Eppendorf, Germany) under the following programs:denaturation at 94 �C for 10 min followed by 30 cycles of 94 �C for30 s, 63 �C for 30 s and 72 �C for 30 s (for lectin); 35 cycles of 94 �Cfor 30 s, 56 �C for 30 s and 72 �C for 30 s (for RRS) and the finalextension was carried out at 72 �C for 3 min. Each extract wasamplified in duplicate assays. In each run four controls were in-cluded, maize and crude soybean oil as negative controls, 0.1%CRM RRS as positive control and blank control.

The amplification fragments were determined using electro-phoresis on 2% agarose gel containing ethidium bromide (0.5 g/mL). A Fast Ruler DNA Ladder Low Range (Fermentas) was usedas a marker.

The agarose gel was visualised in UV transilluminator, and theimages were captured with DOC PRINT system (Vilber Lourmat,USA).

2.4. Real-time PCR

DNA quantification was performed on 7500 Real Time PCR Sys-tem (Applied Biosystems, USA) using the TaqMan Soy 35S GMOdetection kit for the amplification of the soybean lectin (Le 1) genetarget and the p35S target in the same tube. Reactions were carriedout in 96-well microtiter plates in a total volume of 25 ll.

Temperature programme included: initial denaturation during10 min at 95 �C followed by 40 cycles consisting of 95 �C 15 s,60 �C 1 min and 72 �C 31 s. Each sample was amplified in triplicate.

3. Results and discussion

In order to isolate DNA from crude soybean oil obtained fromseed with the different percentage of GMO, two extraction proto-cols were used, CTAB and DNeasy Plant Mini Kit (Qiagen). Thesemethods were chosen as commonly used in the GMO detectionlaboratories. In terms of simplicity and speed the DNeasy PlantMini Kit was easy to use, while the CTAB was the very laboriousand time-consuming method but widely used for food and feed.DNeasy Plant Mini Kit is like as other producer’s commercial kitsbased on the selective adsorption of nucleic acids to a silica-gelmembrane in the presence of high concentrations of chaotropicsalts.

The results of comparative analysis showed that both methodsgave good yield of DNA, which could be explained with the factthat oil was extracted from fine grounded soybean seeds (Table 2).

It is generally agreed that an A260/A280 ratio of 1.8 for DNA isindicative of a pure nucleic acid preparation (Sambrook & Russel,2001). Absorption at 230 nm reflects contamination on the sampleby components such as carbohydrates, peptides, phenols or aro-

th (bp) References

Lipp et al. (2001)

Tengel, Schüßler, Setzke, Balles, and Sprenger-Haußels (2001)

Wurz and Willmund (1997)

Table 2Concentration and purity of DNA obtained with CTAB and DNeasy Plant Mini Kit from oil samples. Values represent average over two replications with standard error.

Oil samples CTAB DNeasy Plant Mini Kit

DNK (ng/ll) A260/A280 A260/A230 DNK (ng/ll) A260/A280 A260/A230

Oil from 0% RR soybean 94.38 ± 0.92 1.73 ± 0.12 0.82 ± 0.15 126.27 ± 0.38 1.80 ± 0.07 0.73 ± 0.09Oil from 1% RR soybean 265.30 ± 1.01 1.38 ± 0.15 0.30 ± 0.06 593.94 ± 0.95 1.94 ± 0.08 1.04 ± 0.09Oil from 5% RR soybean 271.38 ± 0.98 1.46 ± 0.09 0.31 ± 0.11 115.74 ± 0.87 1.72 ± 0.08 0.73 ± 0.08Oil from 10% RR soybean 396.53 ± 0.59 1.14 ± 0.08 0.23 ± 0.06 391.84 ± 0.64 1.92 ± 0.05 1.07 ± 0.04Oil from 100% RR soybean 471.64 ± 2.57 1.45 ± 0.29 0.32 ± 0.18 223.59 ± 0.72 1.86 ± 0.10 1.02 ± 0.11

1074 Z. Nikolic et al. / Food Chemistry 145 (2014) 1072–1075

matic compounds. When the absorption ratio for 260/280 nm isbetween 1.5 and 2.0, and the absorption for 260/230 nm is morethan 1.7, the extracted DNA should be considered as pure DNA.The successful DNA extraction was obtained from all soybean oilsamples. The A260/A280 values and low A260/A230 ratio demonstratethe presence of contaminants in all DNA samples extracted byCTAB method. All samples extracted by DNeasy Plant Mini Kit havehigh A260/A280 values, ranging from 1.72 to 1.94. The low A260/A230

values indicate the presence of carbohydrates or proteins, butusing DNeasy Plant Mini Kit contaminant’s concentration was sig-nificantly reduced. The findings illustrate that the DNA extractionmethods have a significant effect on DNA quality. The similar re-sults were reported by Jasbeer, Son, Mohamad Ghazali, and Cheah,(2009) for the DNA isolated from feed samples. Moreover, the suc-cessful DNA extraction from vegetable oils, enabling the detectionof GMO in these products, was based on using Nucleospin food kitas the most optimal protocol (Costa et al., 2010).

The presence of soybean DNA in oil samples and its applicabilitywas checked by using soybean specific primers for lectin gene.From several primers available in the literature two primers setswere chosen, producing short fragment of 118 bp (GM03/GM04)and longer fragment of 318 bp (Le1/Le6). Using 100 ng of DNAper reaction, the CTAB extracts of DNA were not amplified in allsamples (data not shown). In order to remove the influence ofinhibitory substances, contained mainly in CTAB extracts of DNA,PCR was repeated with diluted samples (50 ng in the reaction)and similar results were obtained. The fragments of 118 bp(Fig. 1) and 318 bp, corresponding to a part of the endogenous lec-tin gene, were amplified in all the samples extracted by DNeasyPlant Mini Kit using 100 ng of DNA in the reaction as well as with50 ng. The DNeasy Plant Mini Kit, similar as Nucleospin food kit,proved to produce amplifiable DNA from refined vegetable oils(Costa et al., 2010a), is based on a silica membrane technology. Thiswork showed that successful DNA extraction from crude oil did notrequire a large amount of oil samples. The quality of isolated DNAfrom crude soybean oil using CTAB method was not sufficient tomake PCR analysis possible, which is case for refined oil, too (Costaet al., 2010b).

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

(bp)

1500

850

400

200

50

118 bp

Fig. 1. Detection of the soybean lectin gene using primers GMO3/GMO4 in soybeanoil samples extracted with CTAB and DNeasy Plant Mini Kit. Line (1) DNA ladder, (2)blank, lines 3–10 CTAB extracts: (3) maize (negative control), (4) 0.1% RRS CRM(positive control), and (5) crude soybean oil, (6–10) soybean oil samples with 0%,1%, 5%, 10% and 100% RR soybean, (11–16) DNeasy Plant Mini Kit extracts (crudesoybean oil, soybean oil samples with 0%, 1%, 5%, 10% and 100% RR soybean) and(17) DNA ladder.

The results pointed out the importance of the DNA extractionprotocol on the oil and other processed food. The presence of dif-ferent inhibitors such as proteins, fats, polysaccharides and othercompounds in DNA extracted from food matrices could affect theamplification step, leading to false-negative results (Corbisieret al., 2007).

For the correct traceability of transgenic materials or whendealing with certified reference materials at a low percentage oftransgenic materials it is important to analyse short fragments(188, 195 or 470 bp) (Bogani, Minunni, Spiriti, Zavaglia, & Tombelli,2009), while Gryson (2010), in a substantial review of methods fordetecting GM DNA in a variety of processed foods, recommendslooking for a maximum of only 150 bp.

Successful GMO detection depends crucially on the quality ofthe extracted sample DNA. The choice of extraction method is of-ten a trade-off between cost, optimal yield of DNA and removalof substances that could influence the PCR reaction (Cankar, Štebih,Dreo, Zel, & Gruden, 2006).

Those samples with positive signal for lectin gene screeningwere analysed for presence of the inserted gene construct in RRsoybean: epsps gene. All expected samples, extracted with DNeasyPlant Mini Kit produced a fragment of 172 bp (Fig. 2). The sensitiv-ity of PCR reaction was checked using as control 0.1% RRS CRM,which gave a visible band.

The amplification of RR soybean by PCR assays using construct-specific primers was achieved for all the extraction containing RRsoybean, except for the CTAB extract of DNA from the oil samplewith 1% RRS.

In order to confirm the qualitative PCR results and to have anestimation the amount of GMO the real-time PCR assays wereconducted.

In all samples with the different percentage of GMO, it was pos-sible to detect and quantify genetically modified organisms. Theresults of GMO quantification also clearly show that, with twoexceptions, all calculations were in line with GMO content in thestarting seed materials (Table 3). The values of quantification inCTAB extracts from 1% and 10% Roundup Ready soybean oils wereunder expected level, probably caused by inhibitors presented inDNA extracts.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

172 bp

(bp)

1000

500

200

50

Fig. 2. Analysis of the presence of RRS in oil samples extracted with CTAB andDNeasy Plant Mini Kit. Line (1) DNA ladder, (1) blank, lines 3–10 CTAB extracts: (3)maize (negative control), (4) 0.1% RRS CRM (positive control), (5) crude soybean oil,(6–10) soybean oil samples with 0%, 1%, 5%, 10% and 100% RR soybean, (11) DNAladder, (12–19) DNeasy Plant Mini Kit extracts: crude soybean oil, soybean oilsamples with 0%, 1%, 5%, 10% and 100% RR soybean, (18) 0.1% RRS CRM (positivecontrol) and (19) maize (negative control), and (20) blank.

Table 3Real-time results for the amplification of oil extracts. Values represent average oversix replications with standard error.

Sample CTAB DNeasy Plant Mini Kit% GMO % GMO

Negative controla NA NA0% NA NA1% 0.52 ± 0.50 1.03 ± 0.475% 6.01 ± 0.10 5.97 ± 0.0510% 1.90 ± 0.59 10.21 ± 0.58100% 98.06 ± 0.13 99.36 ± 0.04

a Negative control – crude soybean oil from market, NA – no detectable ampli-fication LOD = 0.02%, LOQ = 0.1%. R2 = 0.99, slope = �3.42, intercept = 6.94, PCRefficiency = 0.96.

Z. Nikolic et al. / Food Chemistry 145 (2014) 1072–1075 1075

In order to reduce the influence of inhibitors in the extractedsamples, a fourfold dilution series was prepared with water (1:4,1:16, 1:64 and 1:256). All DNA dilutions were run in duplicate.1:16 dilution of the kit extracted DNA resulted in expected quanti-fication, but in CTAB extracted samples inhibitors remained afterdilution. Dilution of the DNA helps to reduce the inhibitor concen-tration and enhance PCR efficiency. However, a lower DNA concen-tration may decrease PCR sensitivity.

Comparing actual and estimated GMO content between twoextraction methods, root mean square deviation for kit is 0.208and for CTAB is 2.127, clearly demonstrate superiority of commer-cial kit over CTAB extraction. Inconsistency in results using CTABextracts, especially at low GMO level, shows that CTAB method isunsuitable for accurate qualitative and quantitative GMO analysisof soybean oil.

The random differences in the reaction conditions due to thesevariations, cause that the standard deviations of the GMO value ofthe samples were relatively high (4–59%). Many manipulationsduring the CTAB extraction require well trained staff, and extrac-tion efficiencies between samples might be altered if the manysamples were extracted at the same time.

The soybean samples used for oil extraction were prepared bymixing GMO and non-GMO soybean on a w/w ratio, so differencesin the genome/weight ratios of the two soybean materials mightexplain the discrepancy. Furthermore, the characteristic of thePCR itself, which does not amplify a target sequence at a 100% effi-cacy, is another important factor that could lead to an underesti-mation of the target copy. In the genetically modified plantsmost of the inserted constructs are present at the level of one copyper haploid genome; it means low copy number targets and it ismore affected by the refining process. The loss of information iseven higher if the plants used for oil production are not 100%genetically modified (Debode et al., 2012).

Since mixtures of GM and non-GM seeds were encountered attrading areas, it is necessary to check for the existence of GMOsin seeds at different points along their path from the field to thefood processing plant (Nikolic, Taški-Ajdukovic, Tatic, & Baleše-vic-Tubic, 2009).

Reliable quantification depends on the efficiency of DNA extrac-tion protocols, which is considered as a critical step in the analysisof DNA extracted from soybean oil. If the oil samples originate fromsoybean seed with varying percentage of RR soybean, it is possibleto monitor the GMO content at the first stage of processing crudeoil.

To our knowledge, this has never been reported before and rep-resents an important accomplishment regarding the traceability ofgenetically modified organisms in oils.

Acknowledgement

This work was supported by the Project No. TR31024 of theMinistry of Education and Science, Republic of Serbia.

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