Semiquantitative analysis of genetically modified maize and soybean in food
María Laura Cazzola
support: MTHA-UNLP (Maestría de Tecnología e Higiene de Alimentos
Keywords: Bt maize, genetically modified organisms, semi-quantitative PCR, RR-soybean, transgenic food.
The aim of this study was to analyze quantitatively the presence of genetically modified organism in food with different composition and degree of processing. Total DNA was extracted by Dellaporta's method and GMO analysis was performed using two consecutives PCR reactions with specie specific primers (IVR and LE), screening primers (35S) and transgen specific primers (CRY and EPSPS). The quantification within the sensitivity establish by the EU was possible only in some foods (ice-cream, flours, soybean isolates and concentrates, starch). Samples with high lipid content or subjected to intense thermal treatments (such as some snacks, mayonnaise, creamy soup) could not be amplified mainly due to the presence of PCR inhibitors. Therefore the method was adequate for identification of food as GM, within the limits establish by EU, only for some Argentinean commercial food products. These findings showed that the develop method was satisfactory only for simple food that were not subject to intense thermal treatments and that do not have high lipid content and that the main limitation of the method is DNA purity.
the last nine years genetically modified plant's growing area has
increased in an exponential way (James, 2004). In
Nevertheless the detection of GMO in foods is very difficult because the matrixes are complex what do not allow an efficient DNA extraction, several PCR inhibitors are present, and in addition DNA suffers alteration during the food processing. The quantitative analysis of GMO in food and ingredients are still more complex, because among its component there are DNAs from different sources and the amount of DNA present in each ingredient will depend on its refinement degree. These facts have opened an intense debate about how the results must be expressed and if it is possible quantify with the limits required. There are also some discussions about the interpretation of the different regulations.
At present, quantitative detection in food has been performed only in some model systems (Yoshimura et al. 2005a; Yoshimura et al. 2005b). The aim of this work was to set up a protocol to semi quantify GMO in foods with different composition and processing degree.
Commercial food samples whose label state the presence of soybean or maize were used. In addition soybean isolates and concentrates were prepared in the laboratory.
Genomic DNA was extracted according to Dellaporta et al. (1983).
conditions were adjusted to quantify with a threshold level of 0.05%.
To this end, different amounts of template and MgCl2,
were assayed, and were finally set in 200 ng template DNA and
the calibration, DNA was extracted from flours prepared mixing different
proportions GM/noGM grains (courtesy of GMO Food Analysis Service
Following ISO 21570 recommendations (ISO, 2002), triplicates were carried out for each curve point. In addition four PCR controls were performed: I- reactives (R): no template, II-positive(+): addition of Bt 176 or RR-DNA; III-negative(-): addition of DNA extracted from non transgenic maize or soybean, IV-extraction (E): addition of the minimal amount of transgenic DNA detectable by the method. At least duplicate PCR of each DNA extracted from food samples were carried out and the presence of PCR's inhibitors (IC) in these preparations was evaluated by addition of 20 ng of transgenic DNA.
The calibration curves obtained using different amounts of DNA from Bt 176 maize or RR soybean, have a lineal range among 0.1 to 20 ng for reactions performed with the primers IVR and 0.1 to 50 ng with the primers LE (Figure 1). Extracted DNA from foods that state in its label maize, such as creamy soup, maize flour, starch, ice-cream, fried potatoes and some snacks are amplified with the primers IVR (Figure 1 and Table 2). In contrast, DNA from other snacks, maize oil or popcorn can not be amplified (Figure 1 and Table 2). In the last group of samples, the control for PCR inhibitors was also negative therefore the purity of DNA was not sufficient. In the case of DNA prepared from food products containing soybean such as isolates, concentrates, flours and ice-cream purity was enough to obtain amplification products with the primers LE (Figure 1 and Table 2). However no amplification was observed for DNA purified from soybean juice, mayonnaise and soy oil. In the case of soybean juice and mayonnaise the lack of amplification was due to the presence of PCR's inhibitors in the DNA preparation (not amplification is observed in the IC reactions) and in the case of oil, whose content of DNA is very low, the extraction yield was not sufficient (in this samples IC was positive).
These results suggest that the DNA extraction method is adequate only for food products with low lipid content and that have not received intense thermal treatments such as flours, isolates, concentrates and ice-cream. Troubles in amplifying complex food samples even using more rigorous DNA extraction methods for example commercial kits has been reported by Peano et al. (2004) and the standardization of extraction methods to obtain pure enough DNA is one of the main challenges to fulfill regulation requirements.
Once the amount of maize or soybean DNA present in the different preparations from food samples was established, in the subsequent PCR, DNA was adjusted to 200 ng, because this amount is necessary to obtain 0.05% sensitivity with the semiquantitative PCR method adjusted in this work. Only positive samples for PCR with the primers IVR and LE were subsequently analyzed by PCR with screening (35S) and identification (CRY and GM07/08) primers. The results obtained for some maize samples are presented in Figure 2. Maize flours and snack sample 3 did not amplify with the primers 35S therefore can be classified as no GM with the threshold level established by the EU (European Council, 2003); in contrast ice-cream and maize starch 2 are positives with the primers 35S therefore should be labeled as GM.
these two samples were negative for transgen-specific reactions with
the primers CRY, therefore the source of the transgene might not be
any of the transgenic maize approved for commercialization in
results obtained for the different maize and soybean contained samples
are summarized in Table 2. Quantification with
the threshold level fixed by EU was possible only in few samples that
are commercialized in
In conclusion, GMO quantification, in the conditions assayed in this work, was only possible for very simple food products that are not subjected to intense thermal treatment and their lipid content is low. The main limitation for the quantitative analysis of food samples was the quality of DNA obtained.
COUNCIL. Regulation (EC) No. 1830/2003., of the European Parliament
and of the Council, concerning the Traceability and Labelling of
Genetically Modified Organisms and the Traceability of Food and
Feed Products Produced from Genetically Modified Organisms and Amending
Directive 2001/18/EC. 15798/1/ 02 REV1. European Council,
GACHET, E.; MARTIN, G.; VIGNEAU, F. and MEYER, G. Detection of genetically modified organisms (GMOs) by PCR: a brief review of methodologies available. Trends in Food Science and Technology, 1999, vol. 9, no. 11-12, p. 380-388. [CrossRef]
GREINER, R.; KONIETZNY, U. and VILLAVICENCIO, A.L.C.H. Qualitative and quantitative detection of genetically modified maize and soy in processed foods sold commercially in Brazil by PCR-based methods. Food Control, 2005, vol. 16, no. 8, p. 753-759. [CrossRef]
21570. Foodstuffs- Methods of analysis for the detection of genetically
modified organisms and derived products- Quantitative nucleic acid
based methods. International Organisation for Standardisation,
MEYER, R. Development and application of DNA analytical methods for the detection of GMOs in food. Food Control, 1999, vol. 10, no. 6, p. 391-399. [CrossRef]
MEYER, R. and JACCAUD, E. Detection of genetically modified soya in processed food products: development and validation of a PCR assay for the specific detection of Glyphosate-Tolerant Soybeans. In: Proceedings of the EURO FOOD CHEM. (IX, 8th -10th September, 1997, Interlaken, Switzerland). Conference, 1997, p. 1, 23-28.
MIRAGLIA, M.; BERDAL, K.; BRERA, C.; CORBISIER, P.; HOLST-JENSEN, A.; KOK, E.; MARVIN, H.; SCHIMMEL, H.; RENTSCH, J.; VAN RIE, J. and ZAGON, J. Detection and traceability of genetically modified organisms in the food production chain. Food and Chemical Toxicology, 2004, vol. 42, no. 7, p. 1157-1180. [CrossRef]
PEANO, C.; SAMSON, M.C.; PALMIERI, L. and GULLI, M. Qualitative and quantitative evaluation of the genomic DNA extracted from GMO and non-GMO foodstuffs with four different extraction methods. Journal of Agricultural and Food Chemistry, 2004, vol. 52, no. 23, p. 6962-6968. [CrossRef]
PIETSCH, K.; WAIBLINGER, H.; BRODMANN, P. and WURZ, A. Screeningverfahren zur Identifizierung "gentechnisch veränderter" pflanzlicher. Lebensmittel. Deutsche Lebensmittel-Rundschau, 1997, vol. 93, no. 2, p. 35-38.
YOSHIMURA, T.; KURIBARA, H.; MATSUOKA, T.; KODAMA, T.; IIDA, M.; WATANABE, T.; AKIYAMA, H.; MAITANI, T.; FURUI, S. and HINO, A. Applicability of the quantification of genetically modified organisms to foods processed from maize and soy. Journal of Agricultural and Food Chemistry, 2005a, vol. 53, no. 6, p. 2052-2059. [CrossRef]
YOSHIMURA, T.; KURIBARA, H.; KODAMA, T.; YAMATA, S.; FUTO, S.; WATANABE, S.; AOKI, N.; IIZUKA, T.; AKIYAMA, H.; MAITANI, T.; NAITO, S. and HINO, A. Comparative studies of the quantification of genetically modified organisms in foods processed from maize and soy using trial producing. Journal of Agricultural and Food Chemistry, 2005b, vol. 53, no. 6, p. 2060-2069. [CrossRef]
Note: Electronic Journal of Biotechnology is not responsible if on-line references cited on manuscripts are not available any more after the date of publication.
Home | Mail to Editor | Search | Archive