Traceability of Transgenic Soybean from Forage Trough Animal Tissue Till the Food Product

Traceability of Transgenic Soybean from Forage Trough Animal Tissue Till the Food Product

LUCRĂRI ŞTIINŢIFICE MEDICINĂ VETERINARĂ VOL. XLVIII(2), 2015, TIMIŞOARA TRACEABILITY OF TRANSGENIC SOYBEAN FROM FORAGE TROUGH ANIMAL TISSUE TILL THE FOOD PRODUCT OANA-MARIA BOLDURA1, C. BALTĂ3, MIRELA AHMADI1, CAMELIA TULCAN1, I. HUȚU1, C. MIRCU1, SORINA POPESCU2 1Banat University of Agricultural Sciences and Veterinary Medicine ―King Mihai I of Romania‖ Timisoara, Faculty of Veterinary Medicine, 300645, Aradului Street, No. 119, Timisoara, Romania 2Banat University of Agricultural Sciences and Veterinary Medicine ―King Mihai I of Romania‖ Timisoara, Faculty of Horticulture and Forestry 3“Vasile Goldis” Western University of Arad, Romania, Institute of Life Sciences E-mail: [email protected] Summary In recent years, there has been a notable concern on the safety of genetically modified (GM) foods/plants, an important and complex area of research, which demands rigorous standards. Molecular methods of GMO detection in food products are based on a short DNA sequence identification. Those sequences can be found in any type of product more or less processed. Even though it was demonstrated by numerous studies that those transgenic sequences will be found in the tissue of any animal that is fed with and were it does not have any metabolic function, there is a lack of studies concerning them crossing in the alimentary products. The European legislation outlines labeling as GMO any food or feed product that contains equal or over 0.9 % GMO material. The presence of remaining DNA sequences in the tissues could easily contribute in reaching and even overcoming of this threshold. Starting from those suppositions, different types of tissues becoming from pigs that were fed with transgenic soybean, were analyzed. GMO detection was accomplished in respect of current specific legislation, following the European standards that were implemented in laboratory, by using PCR type analysis. In the frame of this experiment, sequences of transgenic DNA were identified in fresh but also after a simulation of processing tissues in order to prove the detection all along the technological process. This study emphasizes the necessity of considering those aspects in the process of legally labeling GMO products. Also, there is a need of a better communication for informing the producers and consumers concerning the detection methods and the labeling legislation. Key words: GM soybean, GM food, GM feed, detection, farm pig In recent years, genetically modified (GM) plants, whose DNA has been changed using genetic engineering techniques, are used as foods for human and feeds for farm animals. The majority of GM crops currently produced, like soybean, corn, cotton and canola, have been engineered to enhance agronomic performance by adding transgenic genes for herbicide tolerance and pest resistance. GM soybean is tolerant to the glyphosate family of herbicides by 5 LUCRĂRI ŞTIINŢIFICE MEDICINĂ VETERINARĂ VOL. XLVIII(2), 2015, TIMIŞOARA expressing transgenic DNA that encodes 5-enolpyruvylshikamate-3-phosphate synthase (CP4 EPSPS). Roundup Ready (RR) soybean is the present principal biotech crop. About 90% of the compound feed produced in the EU contains GM soybean. Based on the European food and feed regulation, since 2003 all foods and feeds containing or derived from approved GM products in amounts that pass the 0.9% threshold are compulsory labeled. However, products such as meat, milk, and eggs, that are derived from livestock fed transgenic feeds are exempt from EU-labeling laws (5). The safety of genetically modified organisms regarding the introduced DNA and protein, is based on strong scientific and market regulatory assessments (4, 8, 9, 13) Moreover, the United Nations World Health Organization (WHO) and Food and Agriculture Organization (FAO) have all stated that DNA, is a safe and natural component of food and feed. Highly sensitive detection technologies such as PCR (Polymerase Chain Reaction) can be used to assess the fate of foreign DNA in animal products ( such as meat, milk, and eggs) becoming from farm animals fed with GMO containing forage. The PCR is the most used DNA-based technique, representing the method of choice for GMO detection in most laboratories. Naturally, the digestive process includes the exposure of the gastrointestinal tract to foreign DNA becoming from digested foods or feeds. Ingested food is mechanically disrupted; the DNA is released and cleaved through acid hydrolysis and enzymatic digestion into small DNA sequences and free nucleotides (12). The activity of various phosphatases and deaminases is further affecting the structural integrity of free DNA. It has been shown that plant DNA is progressively degraded through the digestive tract. Over 50% is degraded in the first third of the intestine and 80% is lost when digesta reaches the terminal ileum (11). More over Schubbert et al. (1997) suggested that approximately 0.1% of purified M13 circular phage DNA ingested by mice could be detected as small fragments in white blood cells 2 to 8 h after feeding. The detected DNA was most likely present within the white cells as part of a normal immune system scavenger process. The digestive fate of transgenic DNA was studied since the advent of recombinant DNA methodologies (10, 14) and continues till present time, raising more issues about traceability and precision of detection. The actual trend is to detect small traces of transgenic DNA in different tissues of GM feed animals (1, 2, 3; 6, 15, 16, 19), in attempts to detect the position and the time that passes till the complete disappearing. The overall conclusion is that the transgenic DNA can be detected in organs of animals up to one month but the presence is somehow hazardous, probably carried by body fluids along with any fed becoming exogenous DNA and is not interfering with any metabolic pathways. However, there are no studies designed to follow the sequences of transgenic DNA from tissue trough the processing till the final product. 6 LUCRĂRI ŞTIINŢIFICE MEDICINĂ VETERINARĂ VOL. XLVIII(2), 2015, TIMIŞOARA Materials and methods The biological material was represented by five pigs, noted A, B, C, D, E, grown in a local farm for slaughtering, normally fed with GM soybean containing forage. In the moment of slaughtering the fresh tissue samples were collected: liver, muscle and stomach. Also, a sample of forage was collected and analyzed along with the animal tissues. Certified reference material (CRM) - Roundup Ready™ soybean 1% produced by the Institute for Reference Materials and Measurements (IRMM) was used as control for GMO. Samples preparation For the detection of exogenous DNA in fresh tissue, 50 mg of biological material was sampled. The remaining tissue was minced in sterile condition and boiled in autoclave at 121º C, for 30 minutes. After draying, the tissues were grinded and homogenized. 50 mg of each homogenate were sampled and subjected to DNA extraction. DNA extraction Total DNA was isolated and purified from all samples using CTAB method (ISO 21571, 2005). The quality and quantity of DNA obtained was assessed using NanoDrop8000 spectrophotometer (Thermo Scientific, USA). Primers used in this study Amplificable quality of DNA was confirmed by PCR technique, using pork specific primers, designed for the mitochondrial region 12S rRNA-tRNA Val generating an amplicon of 290 bp length. The primers had the following sequences F 5‘CTACATAAGAATATCCACCACA3‘, R 5‘ACATTGTGGGATCTTCTAGGT3‘.(7) The presence of plant DNA in the samples was assessed by PCR, targeting the chloroplast gene RuBiSco, specific to vegetal genome, with the primersproposed by Rudi et al: CW:5CGTAGCTTCCGGTGGTATCCACGT3', and CX: 5'GGGGCAGGTAAGAAAGGGTTTCGTA3' expected to generate an amplicon of 150 bp. For soybean detection the primers were designed for the lectin gene, with the following sequences: GMO3: 5‘GCCCTCTACTCCACCCCCATCC3‘ and GMO4: 5‘GCCCATCTGCAA GCCTTTTTGTG3‘ and the amplified fragment was 118 bp. Primers HA-nos118-f and HA-nos118-r were used for the detection of the nos terminator, present in the RR soybean having the sequences: nos-f: 5‘GCATGACGTTATTTATGAGATGGG3 ‘and the reverse primer nos-r: 5‘GACACCGCGCGCGATAATTTATCC3‘. The length of the amplified fragment was 118 bp (ISO 21569:2005). PCR amplification The four PCR reactions were run in a final volume of 25 μl using Go Taq Green Master Mix PCR kit from Promega according to producer instructions, 20 7 LUCRĂRI ŞTIINŢIFICE MEDICINĂ VETERINARĂ VOL. XLVIII(2), 2015, TIMIŞOARA pmol of primers and 50 ng/µl DNA template and were performed on a Mastercycler ProS (Eppendorf U.S.) thermalcycler. The amplification program for pork specific primers follows the steps: denaturation 94ºC, 10 min, 35 cycles: denaturation 94ºC, 30 sec, primer annealing 60ºC, 1 min, DNA synthesis 72ºC, 1 min and the final extension 72ºC, 5 min. For RuBiSco primers the PCR program consisted of an initial denaturing step for 3 min at 95°C, followed by 30 cycles of denaturation at 95°C for 20 sec, annealing at 63°C for 45 sec and extension at 72°C for 1 min, with a final step at 72°C for 3 min. The PCR program for lectin primers was: denaturation 95°C - 3 min; 40 cycles: denaturation 95°C -25 sec; primer annealing 62°C - 30 sec, DNA synthesis 72°C - 45 sec; final extension 72°C - 7 min and for the T-nos primers: denaturation 95°C - 3 min; 40 cycles: denaturation 95°C -30 sec; primer annealing 63°C - 30 sec, DNA synthesis 72°C - 30 sec; final extension 72°C - 3 min. The resulting PCR products were separated on 2 % agarose gels in TAE buffer at room temperature at a constant voltage of 100 V for 40 minutes. The PCR products were visualized and photographed under UV light (PhotoDocumentation System, UVP, England). Results and discussions In the first stage of the study, total genomic DNA was isolated and purified from the raw and processed biological samples. DNA of good quality and quantity was obtained and serial dilutions were prepared in the attempt to equalize the genes of interest copies number that may be present in the samples.

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