Ksenija Taski-Ajdukovic, Z. Nikolic,

M. Vujakovic, M. Milosevic,

M. Ignjatov, D. PetrovicNational Laboratory foe Seed Testing, Maksima Gorkog 30, Novi Sad, Serbia

IntroductionThe use of genetically modified organisms (GMO) as food and in food products is becoming more and more widespread. The most cultivated GMO is the Roundup Ready (RR) soybean, which represents the staple constituents of many foods. The addition of soya proteins to processed meat products has significantly

increased in recent years due to the interesting functional and nutritional properties of these vegetable proteins. Soya proteins help to improve technological processes used in the manufacture of meat products and reduce their formulation cost. In addition the demands of consumers for healthier and safer products have also promoted the use of soya proteins in processed meat products as fat replacers (Castro-Rubio et al. 2005).Current EU regulations stipulate that products containing an ingredient of which 0.9% originates from a GM product must be labelled. The law in Serbia is

according to the law in the EU, hence it forbids introduction of

GMO into the environment and demands labelling of food containing more than 0.9% of GMO.The objective of this work was to determine the number of meat products derived from genetically modified soybean in food on the Serbian food market using a conventional qualitative Polymerase Chain Reaction (PCR) assay to detect the presence of RR soya and a real-time PCR to quantify the amount of RR soya present in positive samples.

Materials and methodsFifty processed meat products were gathered randomly from local supermarkets in Novi Sad, Serbia. The Certified Reference Materials (CRM) standards consisting of dried soybean powder with 0, 0.1 and 1% Roundup Ready soybean and

Bt-11 maize produced by the Institute for Research Materials and Measurements (IRMM, Geel, Belgium) were used as positive and negative controls. DNA from samples and reference materials was extracted following

the CTAB precipitation method (Somma, 2004). The concentration and purity

of extracted DNA were measured by absorbance at 260/280 nm in relation to DNA

standard of known concentration (Calf Thymus final concentration of 25 ng/µl).Since genetically modified soybean contains an inserted gene regulated by the 35S promoter, the primers for its amplification were used (Lipp et al. 1999). Primers according to Meyer et al. (1996) were used for identification of soybean DNA. The following reagents were used for duplex PCR amplification: 25 µl of PCR mixture containing 2.5 µl of reaction buffer (Fermentas); MgCl2 1.5 mM, 0.2 mM dNTP; 0.6 µM primers for 35S and 0.1 µM primers for lectin; 1 unit Taq native polymerase (Fermentas) and approx. 100 ng DNA.Amplifications were carried out under the following programs: denaturation at 95 °C for 2 min, followed by 40 cycles at 95 °C for 25 s, 60 °C for 30 s, 68 °C for 45 s, and the final extension was carried out at 68 °C for 10 min. Positive, negative and non-template controls were used in every PCR. PCR products were determined using electrophoresis on a 2% agarose gel

containing ethidium bromide (0.5 g/mL) on the basis of a standard known to be genetically modified. A GeneRuler 50 bp DNA ladder (Fermentas) was used as a size reference.Quantification of the GMO level in positive samples was done by Real-Time PCR according to protocol Taski-Ajdukovic et al. 2006.

Results and discussionHigh quality DNA was extracted from all samples by the CTAB method. The OD260/OD280 of extracted DNA ranged from 1.8-2.0. These DNA samples were used as a template for the PCR analysis.The amplifiability of the DNA extracted from the samples was confirmed using primers for soya specific gene, through visualization of 118 bp amplicons in duplex PCR (Fig. 1). The presence of these amplicons in all tested samples confirmed that the CTAB protocol can be used for DNA extraction and purification from processed meat products. The lectin was detected in all samples, confirming that all samples contained soybean products. These results are in agreement with those presented by Cardarelli et al. (2005), who verified the presence of the lectin gene in all tested sausage samples, and with the results from Fabio at al. (2007), who detected lectin gene in all samples of processed meat products.

The 35S promoter regulates the gene expression of Roundup Ready soybean. Amplification of the 35S promoter resulted in the production of DNA fragment of 195 bp showing the presence of transgenic material in some samples, as

illustrated in Fig. 1, (lines 8, 9, 14 and 15).The results for the fifty samples are compiled in Table 1. The presence of the GMO was demonstrated in 12 cases. The positive samples were mortadella (1), hot-dog (2), salami (3), pate (1), sausages (3), luncheon meat (1) and rolada (1). GM positive soy-containing samples were analyzed by RR soy event-specific Real-

time PCR. All samples contained RR soya below 0.1%. No sample contained RR soy above the 0.9% threshold limit. The results demonstrated the presence of RR soybean in processed

meat products commercially available in Serbia, however, all were below 0.1% and labelling was not necessary.

For additional information, please contact:Dr Ksenija Taski-AjdukovicNational Laboratory for Seed Testing, Maksima Gorkog 30, Novi Sad, SerbiaE-mail address: ksenijat@ifvcns.ns.ac.yu

Monitoring of Roundup Ready Soya in Processed Meat Product on the Serbia

Food Market

Figure 1. Agarose gel electrophoresis of PCR products from processed meat product for analysis of 35S promoter and lectin gene1. DNA ladder; 2. non template control; 3. 0% RR soya; 4. Bt maize; 5- 7. 1%, 0.5% and 0.1% RR soya; 8-19. samples

ReferencesCardarelli, P., Branquinho, M. R., Ferreira, R. T. B., Cruz, F. P., & Gemal, A. L. (2005). Detection of GMO in food products in Brazil: the INCQS experience. Food Control, 16(10), 859-866.

Castro-Rubio, F., Garcia, M. C., Rodriguez, R., & Marina, M. L. (2005).

Simple and Inexpensive Method for the Reliable Determination of Additions of Soybean Proteins in Heat-Processed Meat products: An

Alternative to the AOAC Official Method. Journal of Agricultural Food Chemistry, 53, 220-226.

Fabio, C. A., Brod, F. C. A., & Arisi, A. C. M. (2007). Recombinant DNA in meat additives: Specific detection of Roundup Ready soybean by nested PCR. Journal of the Science of Food and Agriculture 87, 1980–1984

Lipp, M., Brodmann, P., Pietsch, K., Pauwels, J., & Anklman, E. (1999). IUPAC collaborative trial study of a method to detect genetically modified soy beans and maize in dried powder. Journal of Association of Official Analytical Chemists International 82(4), 923-928.

Meyer, R., Chardonnens, F., Hübner, P., & Lüthy, J. (1996). Polymerase Chain Reaction (PCR) in the quality and safety assurance of food: detection of soya in processed meat products. Zeitschrift für Lebensmitteluntersuchung und -Forschung A 203(4), 339-344.

Somma, M. (2004). Extraction and purification of DNA. In M. Querci, M. Jermini, & Van den Eade. The analysis of food samples for the presence of genetically modified organisms (special publication 1.03.114, edition). Ispra: European commission, Joint research centre.

Taški-Ajduković

K., Milošević

M., Nikolić

Z., Vujaković

M. (2006) Testing genetically modified seed of agricultural plants in Serbia and Montenegro. “Biotechnology 2006”, 15-16. February, Scientific Pedagogical Publishing, Č. Budejovice, Czech Repubic ISBN 8085645-53-X, 480-482.

Table 1. Results of GMO analysis in processed meat products

195 bp

118 bp

011cooked ham11010pate366salami122rolada144luncheon meat21313hot-dog31010sausages155mortadella

Presence of 35S promoter

Presence of lectin gene

Number of sampleSamples