Food Chemistry 145 (2014) 1072–1075

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Analytical Methods

Detection of genetically modified soybean in crude soybean oil - ´ a, Maja Ignjatov a, Dušica Jovicˇic´ a, Zorica Nikolic´ a,⇑, Ivana Vasiljevic´ b, Gordana Zdjelar a, Vuk Ðordevic a Dragana Miloševic´ a b

Institute of Field and Vegetable Crops, Maksima Gorkog 30, Novi Sad, Serbia 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 2012 Received in revised form 29 August 2013 Accepted 4 September 2013 Available online 11 September 2013 Keywords: GMO Soybean Crude 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 from soybean seed with a different percentage of GMO seed two extraction methods were used, CTAB and DNeasy 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. Comparing actual and estimated GMO content between two extraction methods, root mean square deviation for kit is 0.208 and for CTAB is 2.127, clearly demonstrated superiority of kit over CTAB extraction. The results of quantification evidently showed that if the oil samples originate from soybean seed with varying percentage of RR, it is possible to monitor the GMO content at the first stage of processing crude oil. Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction The commercial cultivation of genetically modified (GMO) soybean varieties began in 1996, and they became predominant in the major soybean producing countries. Today, around 148 million ha of GMO plants are grown and traded, among which about 71% is soybean (James, 2011). The Roundup Ready soybean (RRS), event GTS 40-3-2, is mainly produced by USA, Argentina and Brazil and exporting in EU and other countries, mostly as soybean meal and crude vegetable oils, among other products. Soybean represents about 60% of total oil consumption and usage of GMO soybean seeds for soybean oil production has been continuously increasing. Soybean oil is the most highly consumed vegetable oil worldwide. It appears in a wide variety of processed foods and in industrial products such as fatty acids, soaps and biodiesel. The European Union (EU) has established the legal basis for the traceability and labelling requirements of genetically modified organisms and GMO derived food and feeds (Regulations (EC) No. 1829/2003, 1830/2003). Furthermore, it covers products destined for industrial processing for uses other than consumption (e.g. in the production of a biofuel). Traceability implies a system to document the history of product of the direction from primary raw materials to the finishing consumable (MacDaniel & Sheridan, 2001). In this sense, traceability is needed for all products, which are tradable.

⇑ Corresponding author. Tel.: +381 21 4898 150; fax: +381 21 421 249. E-mail address: [email protected] (Z. Nikolic´). 0308-8146/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2013.09.017

For labelling of highly processed products in which the GMO protein or DNA may be undetectable, to trace the GMO status of the product traceability is especially useful (Davidson & Bertheau, 2007). There are few reports concerning extraction of DNA and detection of GMO in highly processed food such as oil. The initial report presented that no genetic material can be recovered after the first processing steps of soybean oil, and negative result for all fractions of industrially produced soybean oil (Pauli, Liniger, & Zimmermann, 1998). In opposition to this successful detection of DNA fragments in samples of cold pressed oil, as well as in samples of refined oil, have been reported by Hellebrand, Nagy, and Mörsel (1998). Refining process has influence upon the quality and quantity of DNA, and after the degumming DNA was concentrated in water fraction, no DNA could be amplified in the oil fraction (Gryson et al., 2002). A more recent study comparing four different extraction protocols to recover DNA in soybean oil showed that the choice of the extraction method was a critical parameter to detect a specific DNA fragment by PCR and real-time PCR (Costa, Mafra, Amaral, Beatriz, & Oliveira, 2010a). The other study demonstrated that it is possible to extract trace amounts of amplifiable DNA along a complete industrial soybean oil processing chain mostly based on the use of the commercial kit for DNA extraction (Costa, Mafra, Amaral, & Oliveira, 2010b). DNA could be recovered from distinct types of vegetable oil (soybean, rapeseed, maize and flax) at different stages of the oil refining, even in refined oil at least for high copy DNA targets (Debode, Janssen, & Berbe, 2012). Mentioned authors have been working with oil samples obtained from soybean seed with a high percentage, around 50%

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

or 80% of Roundup Ready soybean. There is no report if it is possible to detect and quantify the GMO if the oil samples were obtained 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 soybean are likely to occur (Bullock & Desquilbet, 2002). The aim of this study was to detect and estimate the quantity of Roundup Ready soybean in crude oil extracted from soybean seed with various percentages of GMO seed using two extraction methods.

2. Materials and methods 2.1. Samples Soybean seed of non GMO variety (Vojvodanka, Institute of Field and Vegetable Crops, Novi Sad) and GMO variety, modification GTS 40-3-2 (Roundup Ready, Monsanto), were used in order to 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 amounts of the ground sample of GMO soybean and non-GMO soybean were weighed and mixed thoroughly. Samples were made in two replicates. The Certified reference materials (CRM) were used as dried soybean powder (GTS 40-3-2) with 0.1% and 0% GMO soybean, developed by the Institute for Reference Materials and Measurements (IRMM, Belgium). Unlabeled crude soybean oil bought from a 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 was stirred overnight at room temperature in order to evaporated nhexane. About 14 ml of crude soybean oil was separated into two tubes and centrifuged at 11,000g for 30 min at 4 °C. After centrifugation, the supernatant was discarded. The DNA was extracted from a pellet on two ways, using the cetyltrimethylammonium bromide (CTAB) method (Querci, Jermini, & Van den Eede, 2004) and DNeasy Plant Mini Kit (Qiagen GmbH). The extractions were done in duplicate assays for each sample. The quality and purity of DNA were analysed by spectrophotometry using BioSpec-nano, Shimadzu (Shimadzu Coorporation, Japan). DNA concentrations were determined by UV absorbance at 260 nm. The purity of the DNA was determined by a ratio of the absorbance at 260, 280 and 230 nm. All samples were diluted with nuclease-free water up to 50 ng/ll. 2.3. Qualitative PCR The sequences of oligonucleotide primers used in this work are presented in Table 1. The primers were synthesized by Metabion International AG (Martinsried, Germany).

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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 containing 0.2 pmol/ll primers for lectin gene and RR soybean and approx. first 100 ng and then 50 ng DNA was used. Amplifications were carried out in a Mastercycler ep gradient S termocycler (Eppendorf, Germany) under the following programs: denaturation at 94 °C for 10 min followed by 30 cycles of 94 °C for 30 s, 63 °C for 30 s and 72 °C for 30 s (for lectin); 35 cycles of 94 °C for 30 s, 56 °C for 30 s and 72 °C for 30 s (for RRS) and the final extension was carried out at 72 °C for 3 min. Each extract was amplified in duplicate assays. In each run four controls were included, maize and crude soybean oil as negative controls, 0.1% CRM RRS as positive control and blank control. The amplification fragments were determined using electrophoresis on 2% agarose gel containing ethidium bromide (0.5 g/ mL). A Fast Ruler DNA Ladder Low Range (Fermentas) was used as a marker. The agarose gel was visualised in UV transilluminator, and the images were captured with DOC PRINT system (Vilber Lourmat, USA). 2.4. Real-time PCR DNA quantification was performed on 7500 Real Time PCR System (Applied Biosystems, USA) using the TaqMan Soy 35S GMO detection kit for the amplification of the soybean lectin (Le 1) gene target and the p35S target in the same tube. Reactions were carried out in 96-well microtiter plates in a total volume of 25 ll. Temperature programme included: initial denaturation during 10 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 from seed with the different percentage of GMO, two extraction protocols were used, CTAB and DNeasy Plant Mini Kit (Qiagen). These methods were chosen as commonly used in the GMO detection laboratories. In terms of simplicity and speed the DNeasy Plant Mini Kit was easy to use, while the CTAB was the very laborious and time-consuming method but widely used for food and feed. DNeasy Plant Mini Kit is like as other producer’s commercial kits based on the selective adsorption of nucleic acids to a silica-gel membrane in the presence of high concentrations of chaotropic salts. The results of comparative analysis showed that both methods gave good yield of DNA, which could be explained with the fact that oil was extracted from fine grounded soybean seeds (Table 2). It is generally agreed that an A260/A280 ratio of 1.8 for DNA is indicative of a pure nucleic acid preparation (Sambrook & Russel, 2001). Absorption at 230 nm reflects contamination on the sample by components such as carbohydrates, peptides, phenols or aro-

Table 1 Oligonucleotide primers. Primer

Sequence (50 –30 )

Fragment length (bp)

References

GM03 GM04

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

118

Lipp et al. (2001)

Le1 Le6

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

318

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

35s-f2 petu-r1

TGA TGT GAT ATC TCC ACT GAC G TGT ATC CCT TGA GCC ATG TTG T

172

Wurz and Willmund (1997)

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

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Table 2 Concentration 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

Oil Oil Oil Oil Oil

from from from from from

CTAB

0% RR soybean 1% RR soybean 5% RR soybean 10% RR soybean 100% RR soybean

DNeasy Plant Mini Kit

DNK (ng/ll)

A260/A280

A260/A230

DNK (ng/ll)

A260/A280

A260/A230

94.38 ± 0.92 265.30 ± 1.01 271.38 ± 0.98 396.53 ± 0.59 471.64 ± 2.57

1.73 ± 0.12 1.38 ± 0.15 1.46 ± 0.09 1.14 ± 0.08 1.45 ± 0.29

0.82 ± 0.15 0.30 ± 0.06 0.31 ± 0.11 0.23 ± 0.06 0.32 ± 0.18

126.27 ± 0.38 593.94 ± 0.95 115.74 ± 0.87 391.84 ± 0.64 223.59 ± 0.72

1.80 ± 0.07 1.94 ± 0.08 1.72 ± 0.08 1.92 ± 0.05 1.86 ± 0.10

0.73 ± 0.09 1.04 ± 0.09 0.73 ± 0.08 1.07 ± 0.04 1.02 ± 0.11

matic compounds. When the absorption ratio for 260/280 nm is between 1.5 and 2.0, and the absorption for 260/230 nm is more than 1.7, the extracted DNA should be considered as pure DNA. The successful DNA extraction was obtained from all soybean oil samples. The A260/A280 values and low A260/A230 ratio demonstrate the presence of contaminants in all DNA samples extracted by CTAB method. All samples extracted by DNeasy Plant Mini Kit have high A260/A280 values, ranging from 1.72 to 1.94. The low A260/A230 values indicate the presence of carbohydrates or proteins, but using DNeasy Plant Mini Kit contaminant’s concentration was significantly reduced. The findings illustrate that the DNA extraction methods have a significant effect on DNA quality. The similar results were reported by Jasbeer, Son, Mohamad Ghazali, and Cheah, (2009) for the DNA isolated from feed samples. Moreover, the successful DNA extraction from vegetable oils, enabling the detection of GMO in these products, was based on using Nucleospin food kit as the most optimal protocol (Costa et al., 2010). The presence of soybean DNA in oil samples and its applicability was checked by using soybean specific primers for lectin gene. From several primers available in the literature two primers sets were chosen, producing short fragment of 118 bp (GM03/GM04) and longer fragment of 318 bp (Le1/Le6). Using 100 ng of DNA per reaction, the CTAB extracts of DNA were not amplified in all samples (data not shown). In order to remove the influence of inhibitory 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 lectin gene, were amplified in all the samples extracted by DNeasy Plant Mini Kit using 100 ng of DNA in the reaction as well as with 50 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. This work showed that successful DNA extraction from crude oil did not require a large amount of oil samples. The quality of isolated DNA from crude soybean oil using CTAB method was not sufficient to make PCR analysis possible, which is case for refined oil, too (Costa et al., 2010b).

The results pointed out the importance of the DNA extraction protocol on the oil and other processed food. The presence of different inhibitors such as proteins, fats, polysaccharides and other compounds in DNA extracted from food matrices could affect the amplification step, leading to false-negative results (Corbisier et al., 2007). For the correct traceability of transgenic materials or when dealing with certified reference materials at a low percentage of transgenic 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 for detecting GM DNA in a variety of processed foods, recommends looking for a maximum of only 150 bp. Successful GMO detection depends crucially on the quality of the extracted sample DNA. The choice of extraction method is often a trade-off between cost, optimal yield of DNA and removal of substances that could influence the PCR reaction (Cankar, Štebih, Dreo, Zˇel, & Gruden, 2006). Those samples with positive signal for lectin gene screening were analysed for presence of the inserted gene construct in RR soybean: epsps gene. All expected samples, extracted with DNeasy Plant Mini Kit produced a fragment of 172 bp (Fig. 2). The sensitivity 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 constructspecific primers was achieved for all the extraction containing RR soybean, except for the CTAB extract of DNA from the oil sample with 1% RRS. In order to confirm the qualitative PCR results and to have an estimation the amount of GMO the real-time PCR assays were conducted. In all samples with the different percentage of GMO, it was possible to detect and quantify genetically modified organisms. The results of GMO quantification also clearly show that, with two exceptions, all calculations were in line with GMO content in the starting seed materials (Table 3). The values of quantification in CTAB extracts from 1% and 10% Roundup Ready soybean oils were under expected level, probably caused by inhibitors presented in DNA extracts.

(bp)

1

2 3

4

5

6 7

8 9 10 11 12 13 14 15 16 17

118 bp

1500

(bp)

850

1000

400

500

200

1 2

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

172 bp 200

50 50

Fig. 1. Detection of the soybean lectin gene using primers GMO3/GMO4 in soybean oil 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 (crude soybean oil, soybean oil samples with 0%, 1%, 5%, 10% and 100% RR soybean) and (17) DNA ladder.

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

Z. Nikolic´ et al. / Food Chemistry 145 (2014) 1072–1075 Table 3 Real-time results for the amplification of oil extracts. Values represent average over six replications with standard error. Sample

CTAB % GMO

DNeasy Plant Mini Kit % GMO

Negative controla 0% 1% 5% 10% 100%

NA NA 0.52 ± 0.50 6.01 ± 0.10 1.90 ± 0.59 98.06 ± 0.13

NA NA 1.03 ± 0.47 5.97 ± 0.05 10.21 ± 0.58 99.36 ± 0.04

a Negative control – crude soybean oil from market, NA – no detectable amplification LOD = 0.02%, LOQ = 0.1%. R2 = 0.99, slope = 3.42, intercept = 6.94, PCR efficiency = 0.96.

In order to reduce the influence of inhibitors in the extracted samples, 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 quantification, but in CTAB extracted samples inhibitors remained after dilution. Dilution of the DNA helps to reduce the inhibitor concentration and enhance PCR efficiency. However, a lower DNA concentration may decrease PCR sensitivity. Comparing actual and estimated GMO content between two extraction methods, root mean square deviation for kit is 0.208 and for CTAB is 2.127, clearly demonstrate superiority of commercial kit over CTAB extraction. Inconsistency in results using CTAB extracts, especially at low GMO level, shows that CTAB method is unsuitable for accurate qualitative and quantitative GMO analysis of soybean oil. The random differences in the reaction conditions due to these variations, cause that the standard deviations of the GMO value of the samples were relatively high (4–59%). Many manipulations during the CTAB extraction require well trained staff, and extraction efficiencies between samples might be altered if the many samples were extracted at the same time. The soybean samples used for oil extraction were prepared by mixing GMO and non-GMO soybean on a w/w ratio, so differences in the genome/weight ratios of the two soybean materials might explain the discrepancy. Furthermore, the characteristic of the PCR itself, which does not amplify a target sequence at a 100% efficacy, is another important factor that could lead to an underestimation of the target copy. In the genetically modified plants most of the inserted constructs are present at the level of one copy per haploid genome; it means low copy number targets and it is more affected by the refining process. The loss of information is even 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 at trading areas, it is necessary to check for the existence of GMOs in seeds at different points along their path from the field to the food processing plant (Nikolic´, Taški-Ajdukovic, Tatic, & Baleševic-Tubic, 2009). Reliable quantification depends on the efficiency of DNA extraction protocols, which is considered as a critical step in the analysis of DNA extracted from soybean oil. If the oil samples originate from soybean seed with varying percentage of RR soybean, it is possible to monitor the GMO content at the first stage of processing crude oil. To our knowledge, this has never been reported before and represents an important accomplishment regarding the traceability of genetically modified organisms in oils.

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