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Presence of genetically modified maize and soy in food products sold commercially in Brazil from 2000 to 2005
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Food Control 19 (2008) 499–505 www.elsevier.com/locate/foodcont
Presence of genetically modified maize and soy in food products sold commercially in Brazil from 2000 to 2005 Ralf Greiner a
a,*
, Ursula Konietzny
b
Centre for Molecular Biology, Federal Research Centre for Nutrition and Food, Haid-und-Neu-Straße 9, D-76131 Karlsruhe, Germany b Waldstraße 5c, D-76706 Dettenheim, Germany Received 21 March 2007; received in revised form 21 May 2007; accepted 25 May 2007
Abstract Qualitative and quantitative polymerase chain reaction-based methods to detect genetically modified soy (RoundupReadyTM soy) and maize (Bt176 MaximizerTM maize, Bt11 maize, MON810 YieldGardTM corn, T25 LibertyLinkTM maize) were applied to processed foods sold commercially in Brazil. From 2000 to 2005, 100 food products containing maize and 100 food products containing soy were analysed every single year. The presence of genetically modified soy increased steadily from 13% in 2000 to 78% in 2005. The number of food products containing genetically modified soy in a proportion above 1.0% on the ingredient level, the threshold for labelling according to Brazilian legislation, increased from 11% in 2000 to 36% in 2005. No clear trend was found within maize containing food products. Eight to eleven percent were shown to consist of material derived from genetically modified maize and 4–6% were found to contain more than 1% of genetically modified maize. Ó 2007 Elsevier Ltd. All rights reserved. Keywords: Detection method; GM maize; GM soy; Polymerase chain reaction; Transgenic food
1. Introduction Gene technology provides a substantial opportunity to alter the composition of crops to match current and emerging food and industrial uses. Many of the genetic improvements in crop plants in the foreseeable future will result from the use of molecular genetic techniques to introduce new genes, modify existing ones and to provide more efficient means to identify specific combinations of genes. World-wide, there are currently 100s of field trials of novel transgenic crop lines, evaluating traits such as herbicide resistance, resistance to insects, fungi and viruses, male sterility and restoration, modified oil, protein and starch composition, pharmaceutical production, production of biodegradable plastics, and stress tolerance.
*
Corresponding author. Tel.: +49 0 721 6625 479; fax: +49 0 721 6625 457. E-mail address: [email protected] (R. Greiner). 0956-7135/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodcont.2007.05.016
Over the last decade, farmers have consistently increased their plantings of transgenic crops by double-digit growth rates every single year since transgenic crops were first commercialised in 1996 (James, 2005). The global area of transgenic crops expanded from 1.5 million hectares in 1996 to 90 million hectares in 2005 (James, 2005; www.transgen.de). There was no change in the major transgenic crops over the years (www.transgen.de). Soybeans continued to be the principal transgenic crop, followed by maize, cotton and canola. The number of countries planting transgenic crops increased from 6 to 21 from 1996 to 2005. The majority of the transgenic crops were grown in the US, Argentina, Canada, Brazil and China. Due to commercialisation of foods or food ingredients derived from genetic engineering, many governments have implemented regulations for the use and labelling of such products. Thus, analytical methods were developed to monitor and verify the presence and amount of genetically modified organisms (GMOs) in agricultural crops and in products derived thereof. So far, detection and
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quantification of GMOs and products derived thereof relies either on DNA detection using the polymerase chain reaction (PCR) technique, or on protein detection using immunological assays such as the enzyme-linked immunosorbent assay (ELISA). Protein detection methods are preferred in the United States and mainly used for the examination of grains and raw materials, whereas DNA detection methods are favoured in Europe. The first official methods that have been validated in ring trials were published in the Swiss Food Manual (Schweizerisches Lebensmittelbuch, 1998) and the German official collection of testing methods in accordance with Article 35 of the German Food Act (Official Collection of Test Methods in accordance with Article 35 LMBG, 1997a, 1997b, 1997c, 1998, 1999, 2001, 2002). However, international standardisation and validation of GMO analysis methods by harmonised and accepted protocols is still in its early phases. The objective of this work was to determine qualitatively and quantitatively the occurrence of products derived from genetically modified soy and maize in foods sold commercially in Brazil. Soy and maize were chosen, because these are the major transgenic crops grown world-wide. Furthermore, quantitative PCR-based detection systems are commercially available for the major transgenic soy and maize events (RoundupReadyTM soy, Bt176 Maximizer maize; Bt11 maize, MON810 Yield Gard corn, T25 LibertyÒ Link maize) cultivated.
Corn, GMOQuant LibertyLinkTM T25 Corn, GMOQuant MaximizerTM Bt176 Corn, and GMOQuant RoundupReadyTM soy were from GeneScan Europe (Freiburg, Germany). PCR-primers were synthesised by Invitrogen (Sa˜o Paulo, Brazil). The WizardTM DNA isolation kit was purchased from Promega (Sa˜o Paulo, Brazil). 2.2. DNA isolation For DNA isolation the WizardTM DNA isolation kit was used according to the instructions of the supplier. 300– 500 mg of food material were carefully mixed with 1000 lL extraction buffer (10 mM Tris–OH, 150 mM NaCl, 2 mM EDTA, 1% (w/v) sodium dodecyl sulfate, 85.15 mM guanidinium hydrochloride, 0.93 mg/mL proteinase K; pH 8.0) and incubated at 65 °C over night. The samples were centrifuged at 13,500g for 10 min and the supernatants were transferred into a new tube. After centrifugation at 13,500g for 10 min 500 lL of the supernatants were mixed well with 1 mL of the WizardÒ-resin and the slurry was pushed gently into a WizardÒ-mini column. The column was washed first with 2 mL isopropanol. After centrifugation at 12,000g for 2 min, the DNA was eluted with 50 lL of pre-warmed (65 °C) 10 mM Tris–OH pH 9.0. After 1 min of incubation at room temperature, the DNA was collected by centrifugation for 2 min at 10,000g. The DNA solutions were stored at 20 °C until further use.
2. Materials and methods 2.3. Qualitative PCR 2.1. Materials Food samples were purchased by chance twice a year (April, October) from three local supermarket chains operating in Sa˜o Paulo as well as Rio de Janeiro, Brazil. GMOQuant Bt11 Corn, GMOQuant YieldGardTM MON810
The sequences of the oligonucleotide primers are given in the Table 1. Amplification was performed in 0.2 ml tubes using 25 lL total reaction volumes containing 1.0 lL of the DNA preparations. The reaction mixture consisted of 10 mM Tris–HCl pH 8.3, 50 mM KCl, 1.5 mM MgCl2,
Table 1 Oligonucleotide primers used for PCR Primer
Primer sequence
Amplicon length [bp]
Reference
Control: Maize
IVR1-F IVR1-R
5 0 -CCG CTG TAT CAC AAG GGC TGG TAC C-3 0 5 0 -GGA GCC CGT GTA GAG CAT GAC GAT C-3 0
226
(Official Collection of Test Methods, 2002)
Soy
GMO3 GMO4
5 0 -GCC CTC TAC TCC ACC CCC ATC C-3 0 5 0 -GCC CAT CTG CAA GCC TTT TTG TG-3 0
118
(Official Collection of Test Methods, 1998)
Specific: Bt176 maize
Cry03 Cry04
5 0 -CTC TCG CCG TTC ATG TCC GT-3 0 5 0 -GGT CAG GCT CAG GCT GAT GT-3 0
211
(Official Collection of Test Methods, 2002)
Bt11 maize
IVS2-2 PAT-B
5 0 -CTG GGA GGC CAA GGT ATC TAA T-3 0 5 0 -GCT GCT GTA GCT GGC CTA ATC T-3 0
189
(Official Collection of Test Methods, 2002)
MON810 maize
VW01 VW03
5 0 -TCG AAG GAC GAA GGA CTC TAA CG-3 0 5 0 -TCC ATC TTT GGG ACC ACT GTC G-3 0
178
(Official Collection of Test Methods, 2002)
T25 maize
T25-F7 T25-R3
5 0 -ATG GTG GAT GGC ATG ATG TTG-3 0 5 0 -TGA GCG AAA CCC TAT AAG AAC CC-3 0
209
(Official Collection of Test Methods, 2002)
RoundupReadyTM soy
p35s-f2 petu-r1
5 0 -TGA TGT GAT ATC TCC ACT GAC G-3 0 5 0 -TGT ATC CCT TGA GCC ATG TTG T-3 0
172
(Official Collection of Test Methods, 1998)
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Table 2 PCR programmes
Initial denaturation Number of cycles Denaturation Annealing Extension Final extension
IVR1-F/IVR1-R
GMO3/GMO4
Cry03/Cry04
IVS2-2/PAT-B
VW01/VW03
T25-F7/T25-R3
p35s-f2/petu-r1
10 min, 95 °C 40 30 s, 95 °C 30 s, 64 °C 30 s, 72 °C 7 min, 72 °C
10 min, 95 °C 35 30 s, 95 °C 30 s, 60 °C 25 s, 72 °C 7 min, 72 °C
10 min, 95 °C 40 30 s, 95 °C 30 s, 64 °C 30 s, 72 °C 7 min, 72 °C
10 min, 95 °C 40 30 s, 95 °C 30 s, 64 °C 30 s, 72 °C 7 min, 72 °C
10 min, 95 °C 40 30 s, 95 °C 30 s, 64 °C 30 s, 72 °C 7 min, 72 °C
10 min, 95 °C 40 30 s, 95 °C 30 s, 64 °C 30 s, 72 °C 7 min, 72 °C
10 min, 95 °C 35 30 s, 95 °C 30 s, 62 °C 25 s, 72 °C 7 min, 72 °C
0.001% (w/v) gelatine, 1.0 lM of each primer, 200 lM of each deoxynucleotide triphosphate and 0.5 units of a Hot-Start DNA polymerase in a Perkin–Elmer GeneAmp PCR System 2400. The PCR programmes are summarised in the Table 2. Amplification products were held at 4 °C until analysed by standard agarose gel electrophoresis followed by ethidium bromide staining. 2.4. Quantitative PCR For quantitative PCR the GMOQuant Bt11 Corn, GMOQuant YieldGardTM MON810 Corn, GMOQuant LibertyLinkTM T25 Corn, GMOQuant MaximizerTM Bt176 Corn, and GMOQuant RoundupReadyTM soy DNA Quantification kits were used on an ABI PRISMTM 5700 Detection System (Applied Biosystems, Weiterstadt, Germany) according to the instructions of the supplier. The kits contain the master mixes for the transgenic event and the taxon, calibration DNA standards, and one tube of certified reference material as a positive control. A volume of 5 lL of the suitably diluted DNA solution was added to 20 lL of master mix. The results of quantitative analysis was expressed as the percentage of GM-DNA copy num-
bers in relation to target taxon specific DNA copy numbers calculated in terms of haploid genomes as recommended by the European Commission 787/2004 (European Commission, 2004). 3. Results Qualitative and quantitative PCR-based methods to detect genetically modified soy (RoundupReadyTM soy) and maize (Bt176 MaximizerTM maize, Bt11 maize, MON810 YieldGardTM maize, T25 LibertyLinkTM maize) was applied to processed foods sold commercially in Brazil. From 2000 to 2005, 100 food products containing maize and 100 food products containing soy were analysed every single year. The food products analysed consisted of local products as well as imported ones. Soy containing products were grouped into flour, bakery products, pasta, vegetarian foods, infant foods, desserts, sweets, soy-based drinks, soy protein isolates, tofu-based foods and instant soups whereas maize containing foods were classified into flour, bakery products, cornflakes, tortilla chips, potent, maize starch, infant foods, desserts, instant soups and maize cobs.
78
52
36
36 29
24
21 18 13
22 16
11
2000
2001
2002
2003
2004
2005
Fig. 1. Percentage of soy containing food samples tested positive for RoundupReadyTM soy in 2000–2005. White bars: percentage of soy containing food sample tested positive for RoundupReadyTM soy; grey bars: percentage of soy containing food samples containing RoundupReadyTM soy in a proportion above 1% on ingredient level; *taken from Greiner et al. (2005).
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3.1. Soy containing foods The presence of genetically modified soy in soy containing food products followed a clear trend over the years (Fig. 1). In 2000, 13% of the soy containing products was shown to consist of material derived from RoundupReadyTM soy. The number of food products containing RoundupReadyTM soy increased steadily from year to year and in 2005, 78% of the analysed food products contained Table 3 Detection of RR soy in soy containing products Products
Number of samples analysed
Number of samples in which RR-DNA was detected 2000*
2001*
2002
2003
2004
2005
Soy flour Bakery products Pasta Vegetarian foods Infant foods Desserts Sweets Soy-based drinks Soy protein isolates Tofu-based foods Instant soups
15 24
6 4
7 6
10 9
9 11
13 15
15 22
10 5
0 0
0 1
0 1
4 2
3 1
8 1
7
0
0
0
0
2
5
11 2 6
0 0 0
1 0 1
1 0 0
0 0 1
3 0 2
5 0 5
3
0
2
0
2
2
3
8
3
3
1
4
6
6
9
0
0
2
3
5
8
Total
100
13
21
24
36
52
78
*
material derived from RoundupReadyTM soy. Quantitative analysis revealed that the number of food products containing RoundupReadyTM soy in a proportion above 1% on the ingredient level, the threshold for labelling according to Brazilian legislation, followed a similar trend (Fig. 1). In 2000, 11% of all food products analysed consisted of RoundupReadyTM soy in a proportion above 1%. This number increased to 36% in 2005. None of these food products have been appropriately labelled. Furthermore, the number of food products containing RoundupReadyTM soy in a proportion of more than 50% increased significantly in 2004 and 2005 (Table 5). Of the food products containing soy only soy flour (40%), tofu-based products (37.5%), and bakery products (16%) have been shown to contain RoundupReadyTM soy in 2000, whereas in 2005 in all groups of soy containing foods with the exception of sweets, material derived from RoundupReadyTM soy was found (Table 3). In 2005, 100% of the soy flours, 92% of the bakery products, 80% of the pastas, 20% of the vegetarian foods, 71% of the infant foods, 45% of the desserts, 83% of the soy-based drinks, 100% of the soy protein isolates, 75% of the tofubased foods and 89% of the instant soups consisted of material derived from RoundupReadyTM soy. 3.2. Maize containing foods Compared to the presence of material derived from genetic engineering in soy containing food products, maize containing food products consist to a far lower extent of such material (Fig. 2). In addition, no clear trend in the presence of genetically modified maize in maize containing food products was found over the years. Eight to eleven percent were shown to consist of material derived from
Greiner et al. (2005).
11
9
9
8
8
6
6
6 5
5
5 4
2000
2001
2002
2003
2004
2005
Fig. 2. Percentage of maize containing food samples tested positive for the transgenic maize events Bt176 Maximizer maize, Bt11 maize, MON810 Yield Gard corn, and T25 LibertyÒ Link maize in 2000–2005. White bars: percentage of maize containing food sample tested positive for the transgenic maize events Bt176 Maximizer maize, Bt11 maize, MON810 Yield Gard corn, and T25 LibertyÒ Link maize; grey bars: percentage of maize containing food samples containing transgenic maize in a proportion above 1% on ingredient level; *taken from Greiner et al. (2005).
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Table 4 Detection of GM maize in maize containing products Products
Maize flour Bakery products Cornflakes Tortilla chips Polenta Maize starch Infant foods Desserts Instant soups Maize cob Total *
Number of samples analysed
Number of samples in which GM maize DNA was detected 2000*
2001*
2002
2003
2004
2005
18
1
2
1
2
2
4
12
0
2
3
4
3
3
14 14
0 5
0 1
1 0
2 0
1 0
1 0
18 6
2 0
4 0
1 0
1 0
2 0
3 0
3
0
0
0
0
0
0
3 4
0 0
0 0
0 0
0 0
0 0
0 0
8
0
0
0
0
0
0
100
8
9
6
9
8
11
Greiner et al. (2005).
genetically modified maize and 4–6% were found to contain more than 1% of genetically modified maize (Fig. 2). In contrast to food products containing genetically modified soy, no shift to higher contents of transgenic material was observed in genetically modified maize containing food products in 2004 and 2005 (Table 5). None of the food products containing genetically modified maize above this threshold level have been appropriately labelled. The vast majority of the genetically modified maize positive food products were not of Brazilian origin, but imported into the country (data not shown). Of the foods containing maize only tortilla chips (36%), polenta (11%), and maize flour (6%) have been shown to contain genetically modified maize in 2000 (Table 4). In respect to the genetically modified maize positive food groups the following changes were observed from 2000 to 2005. From 2001 on material derived from genetically modified maize was also detected in cornflakes and bakery products and from 2002 on, all tortilla chips studies were apparently free of the genetically modified maize events Bt11, Bt176, MON810 and T25. In 2005, 25% of the bakery products, 22% of the maize flours, 17% of the polenta and 7% of the cornflakes consisted of material derived from genetically modified maize. 4. Discussion The qualitative detection of RoundupReadyTM soy and the genetically modified maize events Bt176 Maximizer maize, Bt11 maize, MON810 Yield Gard corn, and T25 LibertyÒ Link maize were based on the official methods for the detection of genetically modified foods of the German Food Act LMBG §35 (Official Collections of Test
503
Methods, 1998, 2002), but the CTAB-based method for DNA extraction was substituted by DNA extraction using a DNA-binding silica resin (WizardTM) as described in the Swiss Food Manual (Schweizerisches Lebensmittelbuch, 1998). The detection systems are construct-specific, since the target sequences of the amplification reactions are part of the newly introduced DNA spanning the boundary of at least two adjacent genetic elements. The WizardTM-system was used in this study, because the crucial point in PCR analysis was identified to be the exclusion of PCR inhibitors (Dickinson, Kroll, & Grant, 1995; Greiner & Jany, 2002). Compared to the classical CTAB-based method, the WizardTM-system was shown to be less time-consuming and to result in better DNA quality, but DNA yields are in general lower (Greiner & Jany, 2002). Appropriate controls were processed to avoid misinterpretation of the data due to false negative or false positive results. Suitability of all DNA preparations for PCR was shown by an independent PCR using either the soybean lectin gene (Official Collection of Methods, 1998) or the maize invertase gene (Official Collection of Test Methods, 2002) as the target. Furthermore, one negative extraction control (substitution of sample with double distilled water) for every set of DNA extractions was processed to detect contamination. For quantification, the so called TaqManTM technique was used. This technique makes use of the 5 0 –3 0 exonuclease activity of the polymerase to generate a template-specific fluorescent signal after hydrolysing an internal probe during each step of the PCR. The parameter measured is the thresholdcycle (CT) where each reaction trespasses a certain fluorescence level. This quantitative PCR method is sufficiently robust to be effective for virtually all DNA-containing matrices and has sufficient sensitivity to distinguish between proportions of a product derived from genetic engineering below and above the threshold level of 1% legal in Brazil (Decreto No 4.680, 2003). The results demonstrate the presence of a broad spectrum of different food products consisting of material derived from genetically modified soybeans and/or genetically modified maize in the Brazilian food market. The presence of genetically modified soy in soy containing food products increased steadily from 13% in 2000 to 78% in 2005, whereas independent of the year, about 10% of the maize containing food products were shown to consist of material derived from genetically modified maize. Cardarelli, Branquinho, Fereira, da Cruz, and Gemal (2005) reported comparable values for the presence of RoundupReadyTM soy in soy containing food products collected from 2001 to 2003 from the Brazilian market. Food samples (20%) consisted of materials derived from RoundupReadyTM soy, but no differentiation by the year was given. In the same time period, 27% of the soy containing food samples was identified as positive for RoundupReadyTM soy in this study. This difference might be due to the differences in the number of food samples collected per year and food group within the two studies. Among the maize containing food samples Cardarelli et al. (2005) did not find
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any product consisting of material derived from the transgenic maize events Bt176 MaximizerTM maize and MON810 YieldGardTM maize. In average, 8% of the maize containing food products in this study collected from 2001 to 2003 was identified positive for the presence of material derived from the transgenic maize events Bt176 MaximizerTM maize, Bt11 maize, MON810 YieldGardTM maize or T25 LibertyLinkTM maize. Besides the higher number of food samples analysed in this study (300 compared to 40 in the study of Cardarelli et al. (2005)), the inclusion of imported products might explain the observed difference. The vast majority of the genetically modified maize positive food products were not of Brazilian origin, but imported into the country. In Brazil as well as in Germany, the prevalence of material derived from genetically modified soy (Brazil: 13–78%, Germany: 10–20% (www.transgen.de)) in food products sold commercially was significantly higher compared to the prevalence of material derived from genetically modified maize (Brazil: 8–11%, Germany: 6–9% (www.transgen.de)). This reflects the more extensive use of soy derived products compared to maize derived products by the food industry as well as the fact that soy was an still is the principal transgenic crop cultivated (James, 2005). Obviously, the presence of material derived from RoundupReadyTM soy in food products was significantly higher in Brazil compared to Germany, whereas the percentage of food products tested positive for transgenic maize was comparable. Both, in Germany and in Brazil the presence of genetically modified soy in soy containing food products increased steadily from 2000 to 2005, whereas the presence of material derived from transgenic maize in maize containing food products remained almost constant. However, the increase in the number of food samples tested positive for the presence of material derived from RoundupReadyTM soy was significantly lower in Germany compared to Brazil. In Germany, 10% of the soy containing food samples were found to contain material derived from RoundupReadyTM soy in 2000 and this percentage doubled to 20% in 2005. In Brazil, a sixfold increase in the presence of RoundupReadyTM soy from 13% in 2000 to 78% in 2005 was observed. In addition, the number of food products
containing RoundupReadyTM soy in a proportion of more than 50% increased significantly in 2004 and 2005 (Table 5). The rapid increase in the prevalence of material derived from RoundupReadyTM soy in food products in Brazil might be due to the legalisation of cultivation of this transgenic soy event in 2003 (Guerrante, 2005). Before 2003, RoundupReadyTMsoy was cultivated illegally on up to 50% of the total area under soybean cultivation in Brazil (www.transgen.de). A significant number of the food products tested positive for transgenic soy or maize, respectively, were above the threshold level of 1% for labelling legal in Brazil, but none of these food products were appropriately labelled. The number of food products containing genetically modified soy in a proportion above 1.0% on the ingredient level increased from 11% in 2000 to 36% in 2005. Four to six percent of the maize containing food products was found to contain more than 1% of genetically modified maize. Germany is facing a different situation. Among the maize containing food products only 0.1–0.2% contained material derived from transgenic maize in levels above 0.9% (threshold value from April 18th, 2004 on) (Regulation (EC) No 1829/2003 of the European Parliament and the Council of the European Union) and 1% (threshold level before April 18th, 2004) (Regulation (EC) No 50/2000 of the European Parliament and the Council of the European Union), respectively (www.transgen.de). Among the soy containing food products even a decrease in the presence of material derived from RoundupReadyTM soy above the threshold level of 0.9% and 1%, respectively, from 2.5% in 2000 to 1% in 2004 and 1.2% in 2005 (www.transgen.de). Among others, these differences may be due to the low prevalence of controls in Brazil compared to Germany. The results clearly show that in Brazil there is an urgent need to monitor and verify the presence and amount of GMOs in agricultural crops and in products derived thereof. The introduction of mandatory regulations regarding labelling of food products containing material derived from transgenic crops (Decreto No 4.680, 2003) has generated a demand for laboratories capable of assuring compliance with the Brazilian legislation.
Table 5 Quantification of transgenic soy and maize in food products sold commercially in Brazil Percentage of foods tested positive for GM soy or maize (%)
Number of samples in which RR-DNA was detected
Number of samples in which GM maize DNA was detected
2000
2001
2002
2003
2004
2005
2000
2001
2002
2003
2004
2005
<1 1–10 10–20 20–30 30–40 40–50 50–60 60–70 70–80 80–90 >90
2 7 2 0 0 1 0 0 0 1 0
3 11 3 1 0 0 2 0 1 0 0
8 9 2 1 0 2 1 0 1 0 0
14 13 4 1 0 0 0 1 2 1 0
23 9 5 2 1 1 2 3 4 2 0
42 10 6 4 1 2 3 2 4 4 0
2 5 1 0 0 0 0 0 0 0 0
3 4 2 0 0 0 0 0 0 0 0
1 4 1 0 0 0 0 0 0 0 0
4 4 1 0 0 0 0 0 0 0 0
4 4 0 0 0 0 0 0 0 0 0
6 3 2 0 0 0 0 0 0 0 0
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Food Control 19 (2008) 499–505 www.elsevier.com/locate/foodcont
Presence of genetically modified maize and soy in food products sold commercially in Brazil from 2000 to 2005 Ralf Greiner a
a,*
, Ursula Konietzny
b
Centre for Molecular Biology, Federal Research Centre for Nutrition and Food, Haid-und-Neu-Straße 9, D-76131 Karlsruhe, Germany b Waldstraße 5c, D-76706 Dettenheim, Germany Received 21 March 2007; received in revised form 21 May 2007; accepted 25 May 2007
Abstract Qualitative and quantitative polymerase chain reaction-based methods to detect genetically modified soy (RoundupReadyTM soy) and maize (Bt176 MaximizerTM maize, Bt11 maize, MON810 YieldGardTM corn, T25 LibertyLinkTM maize) were applied to processed foods sold commercially in Brazil. From 2000 to 2005, 100 food products containing maize and 100 food products containing soy were analysed every single year. The presence of genetically modified soy increased steadily from 13% in 2000 to 78% in 2005. The number of food products containing genetically modified soy in a proportion above 1.0% on the ingredient level, the threshold for labelling according to Brazilian legislation, increased from 11% in 2000 to 36% in 2005. No clear trend was found within maize containing food products. Eight to eleven percent were shown to consist of material derived from genetically modified maize and 4–6% were found to contain more than 1% of genetically modified maize. Ó 2007 Elsevier Ltd. All rights reserved. Keywords: Detection method; GM maize; GM soy; Polymerase chain reaction; Transgenic food
1. Introduction Gene technology provides a substantial opportunity to alter the composition of crops to match current and emerging food and industrial uses. Many of the genetic improvements in crop plants in the foreseeable future will result from the use of molecular genetic techniques to introduce new genes, modify existing ones and to provide more efficient means to identify specific combinations of genes. World-wide, there are currently 100s of field trials of novel transgenic crop lines, evaluating traits such as herbicide resistance, resistance to insects, fungi and viruses, male sterility and restoration, modified oil, protein and starch composition, pharmaceutical production, production of biodegradable plastics, and stress tolerance.
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Corresponding author. Tel.: +49 0 721 6625 479; fax: +49 0 721 6625 457. E-mail address: [email protected] (R. Greiner). 0956-7135/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodcont.2007.05.016
Over the last decade, farmers have consistently increased their plantings of transgenic crops by double-digit growth rates every single year since transgenic crops were first commercialised in 1996 (James, 2005). The global area of transgenic crops expanded from 1.5 million hectares in 1996 to 90 million hectares in 2005 (James, 2005; www.transgen.de). There was no change in the major transgenic crops over the years (www.transgen.de). Soybeans continued to be the principal transgenic crop, followed by maize, cotton and canola. The number of countries planting transgenic crops increased from 6 to 21 from 1996 to 2005. The majority of the transgenic crops were grown in the US, Argentina, Canada, Brazil and China. Due to commercialisation of foods or food ingredients derived from genetic engineering, many governments have implemented regulations for the use and labelling of such products. Thus, analytical methods were developed to monitor and verify the presence and amount of genetically modified organisms (GMOs) in agricultural crops and in products derived thereof. So far, detection and
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quantification of GMOs and products derived thereof relies either on DNA detection using the polymerase chain reaction (PCR) technique, or on protein detection using immunological assays such as the enzyme-linked immunosorbent assay (ELISA). Protein detection methods are preferred in the United States and mainly used for the examination of grains and raw materials, whereas DNA detection methods are favoured in Europe. The first official methods that have been validated in ring trials were published in the Swiss Food Manual (Schweizerisches Lebensmittelbuch, 1998) and the German official collection of testing methods in accordance with Article 35 of the German Food Act (Official Collection of Test Methods in accordance with Article 35 LMBG, 1997a, 1997b, 1997c, 1998, 1999, 2001, 2002). However, international standardisation and validation of GMO analysis methods by harmonised and accepted protocols is still in its early phases. The objective of this work was to determine qualitatively and quantitatively the occurrence of products derived from genetically modified soy and maize in foods sold commercially in Brazil. Soy and maize were chosen, because these are the major transgenic crops grown world-wide. Furthermore, quantitative PCR-based detection systems are commercially available for the major transgenic soy and maize events (RoundupReadyTM soy, Bt176 Maximizer maize; Bt11 maize, MON810 Yield Gard corn, T25 LibertyÒ Link maize) cultivated.
Corn, GMOQuant LibertyLinkTM T25 Corn, GMOQuant MaximizerTM Bt176 Corn, and GMOQuant RoundupReadyTM soy were from GeneScan Europe (Freiburg, Germany). PCR-primers were synthesised by Invitrogen (Sa˜o Paulo, Brazil). The WizardTM DNA isolation kit was purchased from Promega (Sa˜o Paulo, Brazil). 2.2. DNA isolation For DNA isolation the WizardTM DNA isolation kit was used according to the instructions of the supplier. 300– 500 mg of food material were carefully mixed with 1000 lL extraction buffer (10 mM Tris–OH, 150 mM NaCl, 2 mM EDTA, 1% (w/v) sodium dodecyl sulfate, 85.15 mM guanidinium hydrochloride, 0.93 mg/mL proteinase K; pH 8.0) and incubated at 65 °C over night. The samples were centrifuged at 13,500g for 10 min and the supernatants were transferred into a new tube. After centrifugation at 13,500g for 10 min 500 lL of the supernatants were mixed well with 1 mL of the WizardÒ-resin and the slurry was pushed gently into a WizardÒ-mini column. The column was washed first with 2 mL isopropanol. After centrifugation at 12,000g for 2 min, the DNA was eluted with 50 lL of pre-warmed (65 °C) 10 mM Tris–OH pH 9.0. After 1 min of incubation at room temperature, the DNA was collected by centrifugation for 2 min at 10,000g. The DNA solutions were stored at 20 °C until further use.
2. Materials and methods 2.3. Qualitative PCR 2.1. Materials Food samples were purchased by chance twice a year (April, October) from three local supermarket chains operating in Sa˜o Paulo as well as Rio de Janeiro, Brazil. GMOQuant Bt11 Corn, GMOQuant YieldGardTM MON810
The sequences of the oligonucleotide primers are given in the Table 1. Amplification was performed in 0.2 ml tubes using 25 lL total reaction volumes containing 1.0 lL of the DNA preparations. The reaction mixture consisted of 10 mM Tris–HCl pH 8.3, 50 mM KCl, 1.5 mM MgCl2,
Table 1 Oligonucleotide primers used for PCR Primer
Primer sequence
Amplicon length [bp]
Reference
Control: Maize
IVR1-F IVR1-R
5 0 -CCG CTG TAT CAC AAG GGC TGG TAC C-3 0 5 0 -GGA GCC CGT GTA GAG CAT GAC GAT C-3 0
226
(Official Collection of Test Methods, 2002)
Soy
GMO3 GMO4
5 0 -GCC CTC TAC TCC ACC CCC ATC C-3 0 5 0 -GCC CAT CTG CAA GCC TTT TTG TG-3 0
118
(Official Collection of Test Methods, 1998)
Specific: Bt176 maize
Cry03 Cry04
5 0 -CTC TCG CCG TTC ATG TCC GT-3 0 5 0 -GGT CAG GCT CAG GCT GAT GT-3 0
211
(Official Collection of Test Methods, 2002)
Bt11 maize
IVS2-2 PAT-B
5 0 -CTG GGA GGC CAA GGT ATC TAA T-3 0 5 0 -GCT GCT GTA GCT GGC CTA ATC T-3 0
189
(Official Collection of Test Methods, 2002)
MON810 maize
VW01 VW03
5 0 -TCG AAG GAC GAA GGA CTC TAA CG-3 0 5 0 -TCC ATC TTT GGG ACC ACT GTC G-3 0
178
(Official Collection of Test Methods, 2002)
T25 maize
T25-F7 T25-R3
5 0 -ATG GTG GAT GGC ATG ATG TTG-3 0 5 0 -TGA GCG AAA CCC TAT AAG AAC CC-3 0
209
(Official Collection of Test Methods, 2002)
RoundupReadyTM soy
p35s-f2 petu-r1
5 0 -TGA TGT GAT ATC TCC ACT GAC G-3 0 5 0 -TGT ATC CCT TGA GCC ATG TTG T-3 0
172
(Official Collection of Test Methods, 1998)
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Table 2 PCR programmes
Initial denaturation Number of cycles Denaturation Annealing Extension Final extension
IVR1-F/IVR1-R
GMO3/GMO4
Cry03/Cry04
IVS2-2/PAT-B
VW01/VW03
T25-F7/T25-R3
p35s-f2/petu-r1
10 min, 95 °C 40 30 s, 95 °C 30 s, 64 °C 30 s, 72 °C 7 min, 72 °C
10 min, 95 °C 35 30 s, 95 °C 30 s, 60 °C 25 s, 72 °C 7 min, 72 °C
10 min, 95 °C 40 30 s, 95 °C 30 s, 64 °C 30 s, 72 °C 7 min, 72 °C
10 min, 95 °C 40 30 s, 95 °C 30 s, 64 °C 30 s, 72 °C 7 min, 72 °C
10 min, 95 °C 40 30 s, 95 °C 30 s, 64 °C 30 s, 72 °C 7 min, 72 °C
10 min, 95 °C 40 30 s, 95 °C 30 s, 64 °C 30 s, 72 °C 7 min, 72 °C
10 min, 95 °C 35 30 s, 95 °C 30 s, 62 °C 25 s, 72 °C 7 min, 72 °C
0.001% (w/v) gelatine, 1.0 lM of each primer, 200 lM of each deoxynucleotide triphosphate and 0.5 units of a Hot-Start DNA polymerase in a Perkin–Elmer GeneAmp PCR System 2400. The PCR programmes are summarised in the Table 2. Amplification products were held at 4 °C until analysed by standard agarose gel electrophoresis followed by ethidium bromide staining. 2.4. Quantitative PCR For quantitative PCR the GMOQuant Bt11 Corn, GMOQuant YieldGardTM MON810 Corn, GMOQuant LibertyLinkTM T25 Corn, GMOQuant MaximizerTM Bt176 Corn, and GMOQuant RoundupReadyTM soy DNA Quantification kits were used on an ABI PRISMTM 5700 Detection System (Applied Biosystems, Weiterstadt, Germany) according to the instructions of the supplier. The kits contain the master mixes for the transgenic event and the taxon, calibration DNA standards, and one tube of certified reference material as a positive control. A volume of 5 lL of the suitably diluted DNA solution was added to 20 lL of master mix. The results of quantitative analysis was expressed as the percentage of GM-DNA copy num-
bers in relation to target taxon specific DNA copy numbers calculated in terms of haploid genomes as recommended by the European Commission 787/2004 (European Commission, 2004). 3. Results Qualitative and quantitative PCR-based methods to detect genetically modified soy (RoundupReadyTM soy) and maize (Bt176 MaximizerTM maize, Bt11 maize, MON810 YieldGardTM maize, T25 LibertyLinkTM maize) was applied to processed foods sold commercially in Brazil. From 2000 to 2005, 100 food products containing maize and 100 food products containing soy were analysed every single year. The food products analysed consisted of local products as well as imported ones. Soy containing products were grouped into flour, bakery products, pasta, vegetarian foods, infant foods, desserts, sweets, soy-based drinks, soy protein isolates, tofu-based foods and instant soups whereas maize containing foods were classified into flour, bakery products, cornflakes, tortilla chips, potent, maize starch, infant foods, desserts, instant soups and maize cobs.
78
52
36
36 29
24
21 18 13
22 16
11
2000
2001
2002
2003
2004
2005
Fig. 1. Percentage of soy containing food samples tested positive for RoundupReadyTM soy in 2000–2005. White bars: percentage of soy containing food sample tested positive for RoundupReadyTM soy; grey bars: percentage of soy containing food samples containing RoundupReadyTM soy in a proportion above 1% on ingredient level; *taken from Greiner et al. (2005).
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3.1. Soy containing foods The presence of genetically modified soy in soy containing food products followed a clear trend over the years (Fig. 1). In 2000, 13% of the soy containing products was shown to consist of material derived from RoundupReadyTM soy. The number of food products containing RoundupReadyTM soy increased steadily from year to year and in 2005, 78% of the analysed food products contained Table 3 Detection of RR soy in soy containing products Products
Number of samples analysed
Number of samples in which RR-DNA was detected 2000*
2001*
2002
2003
2004
2005
Soy flour Bakery products Pasta Vegetarian foods Infant foods Desserts Sweets Soy-based drinks Soy protein isolates Tofu-based foods Instant soups
15 24
6 4
7 6
10 9
9 11
13 15
15 22
10 5
0 0
0 1
0 1
4 2
3 1
8 1
7
0
0
0
0
2
5
11 2 6
0 0 0
1 0 1
1 0 0
0 0 1
3 0 2
5 0 5
3
0
2
0
2
2
3
8
3
3
1
4
6
6
9
0
0
2
3
5
8
Total
100
13
21
24
36
52
78
*
material derived from RoundupReadyTM soy. Quantitative analysis revealed that the number of food products containing RoundupReadyTM soy in a proportion above 1% on the ingredient level, the threshold for labelling according to Brazilian legislation, followed a similar trend (Fig. 1). In 2000, 11% of all food products analysed consisted of RoundupReadyTM soy in a proportion above 1%. This number increased to 36% in 2005. None of these food products have been appropriately labelled. Furthermore, the number of food products containing RoundupReadyTM soy in a proportion of more than 50% increased significantly in 2004 and 2005 (Table 5). Of the food products containing soy only soy flour (40%), tofu-based products (37.5%), and bakery products (16%) have been shown to contain RoundupReadyTM soy in 2000, whereas in 2005 in all groups of soy containing foods with the exception of sweets, material derived from RoundupReadyTM soy was found (Table 3). In 2005, 100% of the soy flours, 92% of the bakery products, 80% of the pastas, 20% of the vegetarian foods, 71% of the infant foods, 45% of the desserts, 83% of the soy-based drinks, 100% of the soy protein isolates, 75% of the tofubased foods and 89% of the instant soups consisted of material derived from RoundupReadyTM soy. 3.2. Maize containing foods Compared to the presence of material derived from genetic engineering in soy containing food products, maize containing food products consist to a far lower extent of such material (Fig. 2). In addition, no clear trend in the presence of genetically modified maize in maize containing food products was found over the years. Eight to eleven percent were shown to consist of material derived from
Greiner et al. (2005).
11
9
9
8
8
6
6
6 5
5
5 4
2000
2001
2002
2003
2004
2005
Fig. 2. Percentage of maize containing food samples tested positive for the transgenic maize events Bt176 Maximizer maize, Bt11 maize, MON810 Yield Gard corn, and T25 LibertyÒ Link maize in 2000–2005. White bars: percentage of maize containing food sample tested positive for the transgenic maize events Bt176 Maximizer maize, Bt11 maize, MON810 Yield Gard corn, and T25 LibertyÒ Link maize; grey bars: percentage of maize containing food samples containing transgenic maize in a proportion above 1% on ingredient level; *taken from Greiner et al. (2005).
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Table 4 Detection of GM maize in maize containing products Products
Maize flour Bakery products Cornflakes Tortilla chips Polenta Maize starch Infant foods Desserts Instant soups Maize cob Total *
Number of samples analysed
Number of samples in which GM maize DNA was detected 2000*
2001*
2002
2003
2004
2005
18
1
2
1
2
2
4
12
0
2
3
4
3
3
14 14
0 5
0 1
1 0
2 0
1 0
1 0
18 6
2 0
4 0
1 0
1 0
2 0
3 0
3
0
0
0
0
0
0
3 4
0 0
0 0
0 0
0 0
0 0
0 0
8
0
0
0
0
0
0
100
8
9
6
9
8
11
Greiner et al. (2005).
genetically modified maize and 4–6% were found to contain more than 1% of genetically modified maize (Fig. 2). In contrast to food products containing genetically modified soy, no shift to higher contents of transgenic material was observed in genetically modified maize containing food products in 2004 and 2005 (Table 5). None of the food products containing genetically modified maize above this threshold level have been appropriately labelled. The vast majority of the genetically modified maize positive food products were not of Brazilian origin, but imported into the country (data not shown). Of the foods containing maize only tortilla chips (36%), polenta (11%), and maize flour (6%) have been shown to contain genetically modified maize in 2000 (Table 4). In respect to the genetically modified maize positive food groups the following changes were observed from 2000 to 2005. From 2001 on material derived from genetically modified maize was also detected in cornflakes and bakery products and from 2002 on, all tortilla chips studies were apparently free of the genetically modified maize events Bt11, Bt176, MON810 and T25. In 2005, 25% of the bakery products, 22% of the maize flours, 17% of the polenta and 7% of the cornflakes consisted of material derived from genetically modified maize. 4. Discussion The qualitative detection of RoundupReadyTM soy and the genetically modified maize events Bt176 Maximizer maize, Bt11 maize, MON810 Yield Gard corn, and T25 LibertyÒ Link maize were based on the official methods for the detection of genetically modified foods of the German Food Act LMBG §35 (Official Collections of Test
503
Methods, 1998, 2002), but the CTAB-based method for DNA extraction was substituted by DNA extraction using a DNA-binding silica resin (WizardTM) as described in the Swiss Food Manual (Schweizerisches Lebensmittelbuch, 1998). The detection systems are construct-specific, since the target sequences of the amplification reactions are part of the newly introduced DNA spanning the boundary of at least two adjacent genetic elements. The WizardTM-system was used in this study, because the crucial point in PCR analysis was identified to be the exclusion of PCR inhibitors (Dickinson, Kroll, & Grant, 1995; Greiner & Jany, 2002). Compared to the classical CTAB-based method, the WizardTM-system was shown to be less time-consuming and to result in better DNA quality, but DNA yields are in general lower (Greiner & Jany, 2002). Appropriate controls were processed to avoid misinterpretation of the data due to false negative or false positive results. Suitability of all DNA preparations for PCR was shown by an independent PCR using either the soybean lectin gene (Official Collection of Methods, 1998) or the maize invertase gene (Official Collection of Test Methods, 2002) as the target. Furthermore, one negative extraction control (substitution of sample with double distilled water) for every set of DNA extractions was processed to detect contamination. For quantification, the so called TaqManTM technique was used. This technique makes use of the 5 0 –3 0 exonuclease activity of the polymerase to generate a template-specific fluorescent signal after hydrolysing an internal probe during each step of the PCR. The parameter measured is the thresholdcycle (CT) where each reaction trespasses a certain fluorescence level. This quantitative PCR method is sufficiently robust to be effective for virtually all DNA-containing matrices and has sufficient sensitivity to distinguish between proportions of a product derived from genetic engineering below and above the threshold level of 1% legal in Brazil (Decreto No 4.680, 2003). The results demonstrate the presence of a broad spectrum of different food products consisting of material derived from genetically modified soybeans and/or genetically modified maize in the Brazilian food market. The presence of genetically modified soy in soy containing food products increased steadily from 13% in 2000 to 78% in 2005, whereas independent of the year, about 10% of the maize containing food products were shown to consist of material derived from genetically modified maize. Cardarelli, Branquinho, Fereira, da Cruz, and Gemal (2005) reported comparable values for the presence of RoundupReadyTM soy in soy containing food products collected from 2001 to 2003 from the Brazilian market. Food samples (20%) consisted of materials derived from RoundupReadyTM soy, but no differentiation by the year was given. In the same time period, 27% of the soy containing food samples was identified as positive for RoundupReadyTM soy in this study. This difference might be due to the differences in the number of food samples collected per year and food group within the two studies. Among the maize containing food samples Cardarelli et al. (2005) did not find
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any product consisting of material derived from the transgenic maize events Bt176 MaximizerTM maize and MON810 YieldGardTM maize. In average, 8% of the maize containing food products in this study collected from 2001 to 2003 was identified positive for the presence of material derived from the transgenic maize events Bt176 MaximizerTM maize, Bt11 maize, MON810 YieldGardTM maize or T25 LibertyLinkTM maize. Besides the higher number of food samples analysed in this study (300 compared to 40 in the study of Cardarelli et al. (2005)), the inclusion of imported products might explain the observed difference. The vast majority of the genetically modified maize positive food products were not of Brazilian origin, but imported into the country. In Brazil as well as in Germany, the prevalence of material derived from genetically modified soy (Brazil: 13–78%, Germany: 10–20% (www.transgen.de)) in food products sold commercially was significantly higher compared to the prevalence of material derived from genetically modified maize (Brazil: 8–11%, Germany: 6–9% (www.transgen.de)). This reflects the more extensive use of soy derived products compared to maize derived products by the food industry as well as the fact that soy was an still is the principal transgenic crop cultivated (James, 2005). Obviously, the presence of material derived from RoundupReadyTM soy in food products was significantly higher in Brazil compared to Germany, whereas the percentage of food products tested positive for transgenic maize was comparable. Both, in Germany and in Brazil the presence of genetically modified soy in soy containing food products increased steadily from 2000 to 2005, whereas the presence of material derived from transgenic maize in maize containing food products remained almost constant. However, the increase in the number of food samples tested positive for the presence of material derived from RoundupReadyTM soy was significantly lower in Germany compared to Brazil. In Germany, 10% of the soy containing food samples were found to contain material derived from RoundupReadyTM soy in 2000 and this percentage doubled to 20% in 2005. In Brazil, a sixfold increase in the presence of RoundupReadyTM soy from 13% in 2000 to 78% in 2005 was observed. In addition, the number of food products
containing RoundupReadyTM soy in a proportion of more than 50% increased significantly in 2004 and 2005 (Table 5). The rapid increase in the prevalence of material derived from RoundupReadyTM soy in food products in Brazil might be due to the legalisation of cultivation of this transgenic soy event in 2003 (Guerrante, 2005). Before 2003, RoundupReadyTMsoy was cultivated illegally on up to 50% of the total area under soybean cultivation in Brazil (www.transgen.de). A significant number of the food products tested positive for transgenic soy or maize, respectively, were above the threshold level of 1% for labelling legal in Brazil, but none of these food products were appropriately labelled. The number of food products containing genetically modified soy in a proportion above 1.0% on the ingredient level increased from 11% in 2000 to 36% in 2005. Four to six percent of the maize containing food products was found to contain more than 1% of genetically modified maize. Germany is facing a different situation. Among the maize containing food products only 0.1–0.2% contained material derived from transgenic maize in levels above 0.9% (threshold value from April 18th, 2004 on) (Regulation (EC) No 1829/2003 of the European Parliament and the Council of the European Union) and 1% (threshold level before April 18th, 2004) (Regulation (EC) No 50/2000 of the European Parliament and the Council of the European Union), respectively (www.transgen.de). Among the soy containing food products even a decrease in the presence of material derived from RoundupReadyTM soy above the threshold level of 0.9% and 1%, respectively, from 2.5% in 2000 to 1% in 2004 and 1.2% in 2005 (www.transgen.de). Among others, these differences may be due to the low prevalence of controls in Brazil compared to Germany. The results clearly show that in Brazil there is an urgent need to monitor and verify the presence and amount of GMOs in agricultural crops and in products derived thereof. The introduction of mandatory regulations regarding labelling of food products containing material derived from transgenic crops (Decreto No 4.680, 2003) has generated a demand for laboratories capable of assuring compliance with the Brazilian legislation.
Table 5 Quantification of transgenic soy and maize in food products sold commercially in Brazil Percentage of foods tested positive for GM soy or maize (%)
Number of samples in which RR-DNA was detected
Number of samples in which GM maize DNA was detected
2000
2001
2002
2003
2004
2005
2000
2001
2002
2003
2004
2005
<1 1–10 10–20 20–30 30–40 40–50 50–60 60–70 70–80 80–90 >90
2 7 2 0 0 1 0 0 0 1 0
3 11 3 1 0 0 2 0 1 0 0
8 9 2 1 0 2 1 0 1 0 0
14 13 4 1 0 0 0 1 2 1 0
23 9 5 2 1 1 2 3 4 2 0
42 10 6 4 1 2 3 2 4 4 0
2 5 1 0 0 0 0 0 0 0 0
3 4 2 0 0 0 0 0 0 0 0
1 4 1 0 0 0 0 0 0 0 0
4 4 1 0 0 0 0 0 0 0 0
4 4 0 0 0 0 0 0 0 0 0
6 3 2 0 0 0 0 0 0 0 0
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