Microsoft Word - 4-Agra_37236 34 Original Article Biosci. J., Uberlândia, v. 34, n. 1, p. 34-41, Jan./Feb. 2018 REAL-TIME PCR FOR TRACEABILITY AND QUANTIFICATION OF GENETICALLY MODIFIED SEEDS IN LOTS OF NON-TRANSGENIC SOYBEAN PCR EM TEMPO REAL PARA RASTREABILIDADE E QUANTIFICAÇÃO DE SEMENTES GENETICAMENTE MODIFICADOS EM LOTES DE SOJA NÃO TRANSGÊNICA Joelma LEÃO-BUCHIR1,2; Gilberto Vinícius Melo PEREIRA1; André Luís Lopes da SILVA1; Silvana ALBAN1; Maria Carolina ROCHA3; Jossimara POLETTINI4; Vanete THOMAZ-SOCCOL1; Carlos Ricardo SOCCOL1 1. Engenharia de Bioprocessos e Biotecnologia, Centro Politécnico, Universidade Federal do Paraná, Curitiba, PR, Brasil. soccol@ufpr.br; 2. Centro de Biotecnologia, Universidade Eduardo Mondlane, Maputo, Moçambique; 3. Departamento de Hidráulica e Saneamento, Centro Politécnico, Universidade Federal do Paraná, Curitiba, Brasil; 4. Universidade do Oeste Paulista, UNOESTE, Presidente Prudente, Brasil ABSTRACT: The constant presence of genetically modified (GM) soybean in conventional seed lots has become a growing problem for international seed trade. In this context, seed companies have prompted the development of routine tests for accurate genetically modified soybean seeds detection. In this study, a quantitative PCR-based method was standardized in order to detect and quantify mixtures of seeds (i.e. certified seed) or GM grains (i.e. seeds came from field) into samples of non-GM soybean, in a way that soybean lots can be assessed within the standards established by legislation. The method involved the use of p35S-f2/petu-r1 primers targeting CP-4 enolpyruvylshikimate-3-phosphate synthase (cp4-epsps) gene (i.e. that confers herbicide tolerance in Roundup ReadyTM (RR)) for real-time PCR detection and quantification through mericon Quant GMO Detection Assay. The results revealed the method efficiency to detect and quantify the presence of even one soybean seed in batch used for routine evaluation of GM seeds. In addition, it was possible to detect of up to 0.1% of transgenic DNA relative to the soybean grains content. Thus, the sensitive GMO quantitative approach described in this study will provide support in supervising activities, and facilitate the process and control of GM soybean. KEYWORDS: Roundup Ready® soybean. GMP. Glycine max. Cp4-epsps gene. INTRODUCTION Concerns about the intrinsic and extrinsic quality attributes in foods have been intensified due to the entry of genetically modified crops in the global consumer market (SIEW-PING et al., 2011). Many of the current debates on agricultural biotechnology have focused on the potential risks of genetically modified (GM) crops for human health. Some of the health risks pertinent to unapproved genetically modified food and concerned consumers include antibiotic resistance, allergenicity, nutritional changes and the formation of toxins (MAGHARI; ARDEKANI, 2011). Thus, large soybean importing countries have internally reached an agreement to satisfy the demands of the consumers. Among these demands is the introduction of mandatory labeling of products containing levels of genetically modified organisms above the allowed limits, without thereby restricting imports of soybean complex (VILJOEN et al., 2006). For instance, the labeling thresholds are defined as 0.9% in the European Union (EUROPEAN COMMISSION, 2003), 1% in Brazil (DE MIRANDA et al., 2005), 3% in South Korea (Notification No.2000-31, 2000), and 5% in Japan (MATSUOKA, 1999). To fulfill labeling requirements, several analytical methods have been used to detect genetically modified crops. Divided in protein-based assays, e.g., ELISA test, provide only quality results about specific proteins contained in the genetically modified material, and DNA-based methodologies, which can be divided into qualitative (conventional PCR) or quantitative (real time PCR) analyses (MAGHARI; ARDEKANI, 2011). The establishment of an adequate, safe and economical method for detection, identification and quantification of GM seeds in conventional batch remains a challenge. Recognizing the importance of new markets and genetically modified products, seed technology must ensure the genetic purity of new biotechnology products, in order to avoid legal proceedings due to incorrect identification of varieties. In this context, the objective of this study was to apply a quantitative PCR-based method for Received: 10/01/17 Accepted: 05/12/17 35 Real-time PCR... LEÃO-BUCHIR, J. et al Biosci. J., Uberlândia, v. 34, n. 1, p. 34-41, Jan./Feb. 2018 detecting and quantifying mixtures of seeds or GM grains into samples of non-GM soybean, in a way that soybean lots can be assessed within the standards established by legislation. MATERIAL AND METHODS Sample material and contamination experiment Samples of GM and conventional soybean seeds (i.e. certified seed) and grains (i.e. seeds came from field) were used in this study. The cultivar used in this research were GM soybean (P98Y30 RR) with the Certified Reference GM-soybean materials were acquired at Pioneer Hi-Bread International, Inc. (USA), while the Certified Reference pure soybean (cv. BRS1010 IPRO) materials (non-GM) were provided by the Department of Agriculture and Supply of Paraná State, Brazil. The sampling procedure was performed in the Laboratory of Products Classification of Paraná (CLASPAR, Curitiba, Brazil) in accordance with the standards established by the International Seed Testing Association (PINTO et al., 2011). The experimental samples were prepared in order to detect contamination of GM seeds within non-GM soybean seed lots. Portions of 500 g of non-GM soybean seed were individually contaminated with 2, 3, 5, 6, 9, 10, 15 and 20 units of GM soybean seeds. The same procedure was applied for soybean grains, except using portions of 100 g of non-GM soybean and contamination levels of 1, 2, 3, 5, 10, 11, 13 and 14 units of GM soybean grains. Each contamination procedure was carried out in triplicate. DNA extraction The samples were transported for the Laboratory of Biotechnological Processes, at Federal University of Paraná, Curitiba, Brazil, for further analysis. Genomic DNA was isolated of each contamination experiment using the Cetyl trimethylammonium bromide (CTAB) method (GREINER; KONIETZNY, 2008; Pinto et al., 2011). The integrity and concentration of the extracted DNA was measured by electrophoresis and spectrophotometer absorbance at 260 and 280 nm wavelength, respectively. Qualitative PCR detection of lectin and GM gene sequences The primers GMO3 (5´- GCCCTCTACTCCACCCCCATCC-3´) and GMO4 (5´-GCCCATCTGCAAGCCTTTTTGTG-3´) were used as control to amplify endogenous lectin gene of soybean according with method described by Greiner & Konietzy (2008). The primers p35S-f2 (5´-TGATGTGATATCTCCACTGACG-3´) and petu-r1 (5´-TGTATCCCTTGAGCCATGTTG-3´) targeting CP-4 5-enolpyruvylshikimate-3-phosphate synthase (cp4-epsps) gene that confers herbicide tolerance gene in RR soybean (SIEW-PING et al., 2011) were used for the detection of GM soybean. The PCR was performed using Veriti Thermal Cycler (Applied Biosystems) in a total reaction mixture volume of 10 µL, containing: 1× PCR buffer, 1.5 mM MgCl2, 0.2 mM dNTPs, 1.5 unit of Taq DNA polymerase, 3 µL of DNA extract and 0.5 µM of GMO3/GMO4 (lectin gene reaction) or 0.8 µM of p35S-f2/petu-r1 (RR soybean gene reaction). For GMO3/GMO4 primers amplification, the following cycle program was used: denaturation at 95°C for 5 min followed by 35 cycles of 95°C for 30 s, 61°C for 30 s, and 72 °C for 1 min; final extension at 72°C for 3 min. The cycle program for primers p35S-f2/petu-r1 was: denaturation at 95˚C for 5 min, followed by 35 cycles of 95˚C for 30 s, 61˚C for 30 s and 72˚C for 30 s; final extension at 72˚C for 3 min. The PCR products were separated by electrophoresis at 80V for 50 min in a 1.5% agarose gel, 1X TBE buffer and stained with ethidium bromide. The PCR products were visualized in a UV-trans illuminator and the images photographed with a digital camera (Loccus biotecnologia L.PIX System). Real-time PCR The real-time PCR for quantitative detection of GM content and control lectin gene was carried out in a 7500 Fast Real-Time PCR System (Applied Biosystems) using Mericon Quant GMO Detection Assays kit (Qiagen). A volume of 4.8 µ L of prepared samples, positive and negative controls, as well as the provided standards dilutions were added to the reagents according to manufacturer’s instructions. PCR amplification were carried out with an initial denaturation for 5 min at 95°C, followed by 45 cycles of 95 ̊C for 15 s, 60 ̊C for 30 s and 72 ̊C for 10 s. The results of the amplification curve and threshold cycle values (Ct) were analyzed using 7500 Software Version 2.05 (Applied Biosystems). The threshold cycle number (CT) was used for quantification of GMO in lots of soybean seeds and grains, using the ddCT method (i.e. the algorithm for the analysis of quantitative real-time PCR (qRT-PCR)). The mean of CT from prepared samples was compared to the standard curve Ct means. All reactions were performed in duplicate and no template controls were included in each run. 36 Real-time PCR... LEÃO-BUCHIR, J. et al Biosci. J., Uberlândia, v. 34, n. 1, p. 34-41, Jan./Feb. 2018 The results are expressed in percentage by comparing the samples with the standard. RESULTS AND DISCUSSION DNA extraction and qualitative PCR In this study, the CTAB method was efficient for extracting DNA from soybean seed and grain varieties, with good quality and appropriate quantity as demonstrated in Table 1. An important point concerning PCR analysis is the presence of PCR inhibitors in the amplification reactions, which could lead to false-negative results (MAFRA et al., 2008). Thus, it becomes crucial to standardize protocols for the extraction of DNA from food samples to avoid PCR inhibitors due to DNA extraction inadequate. The CTAB is a widely used method of DNA extraction and described in the official methods for the detection of GM foods of the German Food Act LMBG §35 (Greiner et al., 2005). Table 1. Results of the isolated DNA in soybean seeds and grains (cv. P98Y30 RR (GM = genetically modified) and cv. BRS1010 IPRO (non-GM = non-genetically modified)) and qualitative PCR with each primer pair evaluated, being (GMO3/GMO4) for lectin gene used as endogenous reference gene (i.e. to confirm the presence of soybean residues in the samples) and (p35s-f2/petu-r1) for cp4 epsps gene that confers herbicide tolerance in Roundup ReadyTM (RR). SS = Soybean certified seeds and Cc = Soybean grains (i.e. seeds came from field). Absorbance (nm) Primers pairs and amplified product (bp) 260 280 [DNA] (ng/µ l)) Sample Designation GMO3/GMO4 p35s-f2/petu-r1 0.067 0.039 167.5 C+ GM + (118) + (172) 0.032 0.027 80 C- Non-GM + (118) - (172) 0.032 0.016 80 SS2 GM + (118) + (172) 0.094 0.061 235 SS3 GM + (118) + (172) 0.064 0.044 160 SS5 GM + (118) + (172) 0.098 0.062 245 SS6 GM + (118) + (172) 0.062 0.04 155 SS9 GM + (118) + (172) 0.024 0.004 60 SS10 GM + (118) + (172) 0.072 0.04 180 SS15 GM + (118) + (172) 0.027 0.018 67.5 SS20 GM + (118) + (172) 0.009 0.005 22.5 Cc1 GM + (118) + (172) 0.010 0.004 25 Cc2 GM + (118) + (172) 0.018 0.006 45 Cc3 GM + (118) + (172) 0.018 0.022 45 Cc5 GM + (118) + (172) 0.054 0.059 135 Cc10 GM + (118) + (172) 0.076 0.044 190 Cc11 GM + (118) + (172) 0.015 0.019 37.5 Cc13 GM + (118) + (172) 0.012 0.005 30 Cc14 GM + (118) + (172) In the qualitative PCR assay, the use of GMO3/GMO4 primers produced a fragment expected of 118 bp from the lectin gene in all analyzed samples (GREINER; KONIETZY 2008). A method for detection of specific species, such as the soybean lectin gene, is necessary to discriminate between negative and positive results due to inhibition in the amplification. In addition to control PCR, specific detection with the primers p35s- f2/petu-r1 yielded a fragment of 172 bp complementary to the CP-4 5- enolpyruvylshikimate-3-phosphate synthase (cp4- epsps) gene that confers herbicide tolerance gene in RR soybean (PINTO et al., 2011). The specificity of this reaction was verified in our study as no amplification product was obtained in parallel experiments using wild-type soybean. A contamination dilution series of GM soybean into lots of wild-type showed that the target fragment was detected from all of the tested levels (Table 1), which indicates the efficiency of the event-specific PCR assay used in this study for detection of transgenic soybean. 37 Real-time PCR... LEÃO-BUCHIR, J. et al Biosci. J., Uberlândia, v. 34, n. 1, p. 34-41, Jan./Feb. 2018 Real-time PCR A quantitative PCR-based method was applied in order to control maximum limits for GM in lots of soybean grains and seeds. The detection of lectin gene (control reaction) gave an average Ct value of 23.00 cycles for seeds (R2 = 0.992) (Table 2) and 24.00 cycles for grains (R2 = 0.999) (Table 3). Table 2. Threshold cycle values (Ct) and copy number of lectin and cp-4 epsps genes from soybean certified seeds (SS). C+ = positive control and C- = negative control. Mericon Assay samples Lectin cp-4 epsps Ct Copy number Ct Copy number STD1 1 21.589 81.920 28.721 10.420 STD 2 24.565 10.420 31.008 1280 STD 3 27.22 1280 33.727 160 STD 4 30.64 160 37.949 20 C+ 25.242 3,082.271 36.034 25.813 C- _ _ _ _ SS22 25.389 2,090.444 36.314 1.50 SS3 25.027 3,073.221 35.5 2.17 SS5 24.052 4,683.718 35.364 464 SS6 22.907 4,806.966 35.221 544 SS9 22.202 5,948.167 34.901 5,444 SS10 22.187 10,161.592 33.793 15,311 SS15 21.33 25,345.625 33.585 119,285 SS20 21.138 67,879.562 32.063 567,975 1 Sample with concentration or with copy number known and these samples were used to build a standard curve. 2 The number after the SS means the number of GM seeds intentionally contaminated in a batch of 500 g of non-GM seeds. Table 3. Threshold cycle values (Ct) and copy number of lectin and cp-4 epsps genes from soybean grains (Cc). C+ = positive control and C- = negative control. Mericon Assay Samples Lectin cp-4 epsps Ct Copy number Ct Copy number STD1 1 22.93 81.920 28.733 10.420 STD 2 25.816 10.420 31.737 1280 STD 3 28.897 1280 34.564 160 STD 4 32.034 160 37.888 20 C+ 26.427 3,207.507 37.907 25.813 C- _ _ _ _ Cc 12 27.572 2,090.444 40 32.65 Cc 2 27.092 3,073.221 40 51.19 Cc 3 26.702 4,683.718 38.816 65.051 Cc 5 26.461 4,806.966 37.227 75.810 Cc 10 26.136 10,161.592 36.725 233.918 Cc 11 25.294 10,345.625 30.934 468.491 Cc 13 21.087 120,345.625 30.712 993.081 Cc 14 20.488 159,835.344 28.389 1,430.225 1 Sample with concentration or with copy number known and these samples were used to build a standard curve. 2 The number after the Cc means the number of GM grains intentionally contaminated in a batch of 100 g of non-GM grains. 38 Real-time PCR... LEÃO-BUCHIR, J. et al Biosci. J., Uberlândia, v. 34, n. 1, p. 34-41, Jan./Feb. 2018 For cp-4 epsps gene amplification, that correlated with the amount of GM content present in each lot, the Ct value was 34.00 cycles for seeds (R2 = 0.952) (Table 2) and 34.00 cycles for grain (R2 = 0.965) (Table 3). For all reactions, PCR efficiency over 98% was observed which indicates the suitability of this method for detection/quantification of GMO in lots of soybean seeds and grains. These results are similar to those observed by Siew-Ping et al. (2011) in quantifying cp-4 epsps soybean gene in others food matrixes. Table 4 shows the quantification results of the transgenic DNA gene content in each soybean lot intentionally contaminated with GM seeds and grains. Table 4. Quantitative analysis of the Roundup ReadyTM soybean certified seeds (SS) and grains (Cc). % GM = Percentage of detected genetically modified material and C+ = positive control. Sample % GM % GM Soybean seed Soybean grain C+ 0.99 C+ 0.99 SS21 0.007 Cc 1 0.1 SS3 0.007 Cc 2 0.1 SS5 0.009 Cc 3 1.3 SS6 0.01 Cc 5 1.5 SS9 0.09 Cc 10 2.3 SS10 0.2 Cc 11 4.5 SS15 0.5 Cc 13 8.2 SS20 0.8 Cc 14 8.9 1 The number after the SS means the number of GM seeds intentionally contaminated in a batch of 500 g of non-GM seeds. The good linearity between transgenic DNA quantities and fluorescence values (Ct) confirm that the assay is well suited to quantitative measurements. In relation to seed evaluation, the quantitative PCR assay enabled the detection of even one soybean grain or seed in batch used for routine evaluation of GM soybean, providing a rigorous process in detecting GMO soybean seeds. For soybean grains mensuration, the method was able to detect the presence of up to 0.1% of transgenic DNA. The threshold of 0.1% will be suitable for practical detection of GM soybean grain materials. This study gave an insight of the occurrence of different levels of transgenic DNA as an alternative way for tracing GMO in lots of seeds and grains. In relation to seeds, the method enables the detection of even one soybean seed in batch used for routine evaluation of GM soybean. Thus, the sensitive GMO quantitative approach described in this study will provide support in supervisory activities, facilitating the process and control of GM soybean. ACKNOWLEDGEMENTS The authors are grateful the CB-UEM and Rita Levi-Montalcini Foundation for financial support and CNPq for financial support and master scholarship of the first author. RESUMO: A constante presença da soja geneticamente modificada (GM) em lotes de sementes convencionais têm se tornado um grande problema para o comércio internacional de sementes. Neste contexto, as empresas de sementes estão em busca de testes de rotina extremamente precisos para a detecção de sementes de soja geneticamente modificadas. Neste estudo, um método baseado em PCR quantitativo foi padronizado para detectar e quantificar misturas de sementes (i.e. sementes certificadas) ou grãos geneticamente modificados (i.e. sementes oriundas do campo) dentro de lotes de soja não transgênica, de um modo que os lotes de soja possam ser avaliados dentro dos parâmetros estabelecidos pela legislação. O método envolveu o uso dos iniciadores p35S-f2/petu-r1 alvejando o gene CP-4 5-enolpiruvil-shikimato-3- fosfato sintase (cp4-epsps) (i.e. que confere a tolerância ao herbicida Roundup Ready® (RR)) para detecção e quantificação em PCR de tempo real via Ensaio de detecção Mericon Quant GMO. Os resultados revelaram um método eficiente para detectar e quantificar a presença de até mesmo uma única semente de soja no lote usado para a avaliação de rotina de sementes geneticamente modificadas. Adicionalmente, foi possível detectar até 0,1% de DNA transgênico relativo ao conteúdo de grãos de soja. Dessa forma, uma abordagem quantitativa sensível à soja geneticamente modificada foi descrita 39 Real-time PCR... LEÃO-BUCHIR, J. et al Biosci. J., Uberlândia, v. 34, n. 1, p. 34-41, Jan./Feb. 2018 nesse estudo e poderá fornecer suporte em atividades de supervisão, além de facilitar o processo de controle da soja geneticamente modificada. PALAVRAS-CHAVE: Soja Roundup Ready®. Planta geneticamente modificada. PCR em tempo real. Glycine max. Gene cp4-epsps. REFERENCES ABDULLAH, T.; RADU, S.; HASSAN, Z.; HASHIM, J. K. Detection of genetically modified soy in processed foods sold commercially in Malaysia by PCR – based method. Food Chemistry, Amsterdam, v. 98, p. 575- 579, 2006. https://doi.org/10.1016/j.foodchem.2005.07.035 AERNI, P. Stakeholder attitudes towards the risks and benefits of genetically modified crops in South Africa. Environmental Science and Policy, Netherlands, v. 8, p. 464-476, 2005. https://doi.org/10.1016/j.envsci.2005.07.001 ALEXANDER, T. W.; REUTER, T.; AULRICH, K.; SHARMA, R.; OKINE, E. K.; DIXON, W. T.; McALLISTER, T. A. A review of the detection and fate on novel plant molecules derived from biotechnology in livestock production. Animal Feed Science and Technology, Amsterdam, v. 133, p. 31-62, 2007. https://doi.org/10.1016/j.anifeedsci.2006.08.003 ASSOCIAÇÃO BRASILEIRA DOS PRODUTORES DE MILHO (ABRAMILHO). Disponível em: ˂http://www.abramilho.org.br/noticias.php?cod=1405˃. Acesso em: 07/2012. BONFINI, L.; HEINZE, P.; KAY, S.; VAN DEN EEDE, G. Review of GMO detection and quantification techniques. European Commission, Italy, 2001. BRASIL. Decreto no 4689, de 24 de abril de 2003. Regulamenta o direito à informação, assegurado pela Lei no 8078 de 11 de Setembro de 1990, quanto aos alimentos e ingredientes alimentares destinados ao consumo humano ou animal que contenham ou sejam produzidas a partir de organismos geneticamente modificados, sem prejuízo do cumprimento das demais normas aplicáveis. Diário Oficial da República Federativa do Brasil, Brasília, DF, 24 abr. 2003. Disponível em: Acesso em Junho de 2012. CENTRO DE INFORMAÇÃO DE BIOTECNOLOGIA (CIB). Disponível em: ˂ http://cibpt.org/inicio/index.php/cib/comunicados/2012/138-culturas-transgenicas-no- mundo-16-7-milhoes-de- agricultores-cultivaram-mais-8-em-2011˃. Acesso em: 07/2012. CHEN, Y.; WANG, Y.; GE, Y.; XU, B. Degradation of endogenous and exogenous genes of roundup-ready soybean during food processing. Journal of Agricultural and Food Chemistry, Washington DC, v. 53, p. 10239-10243, 2005. https://doi.org/10.1021/jf0519820 DE MIRANDA, D. M.; TILLMANN, M. A. A.; BALERINI, F.; VILLELA, F. A. Bioensaio na detecção e quantificação de sementes de soja geneticamente modificada resistente ao glifosato. Revista Brasileira de Sementes, Londrina, v. 27, n.1, p. 93-103, 2005. https://doi.org/10.1590/S0101-31222005000100012 DINON, A. Z.; TREML, D.; de MELLO, C. S.; ARISI, A. C. M. Monitoring of GMO in Brazilian processed meat and soy-based products from 2007 to 2008. Journal of Food Composition and Analysis, Amsterdam, v. 23, p. 226-229, 2010. https://doi.org/10.1016/j.jfca.2009.12.002 EUROPEAN NETWORK OF GMO LABORATORIES (ENGL). Definition of minimum performance requirements for analytical methods of GMO testing, Community Reference Laboratory, p.8, 2008. 40 Real-time PCR... LEÃO-BUCHIR, J. et al Biosci. J., Uberlândia, v. 34, n. 1, p. 34-41, Jan./Feb. 2018 FERREIRA, R. T. B.; BRANQUINHO, M. R.; CARDARELLI-LEITE, P. Soja geneticamente modificada em alimentos contendo farinha e preparados à base de farinha de trigo. Detecção e adequação à legislação de rotulagem. Brazilian Journal of Food Technology, Campinas, v. 12, n. 3, p. 241-248, 2009. https://doi.org/10.4260/BJFT2009800900018 GACHET, E.; MARTIN, G. G.; VIGNEAU, F.; MEYER, G. Detection of genetically modified organism (OGMs) by PCR. A brief review of methodology available. Trends in Food Science and Technology, Amsterdam v. 9, p. 380-388, 1999. https://doi.org/10.1016/S0924-2244(99)00002-3 GREINER, R.; KONIETZNY, U.; VILLAVICENCIO, A. L. C. H. Qualitative and quantitative detection of genetically modified maize and soy in processed food sold commercially in Brazil by PCR-based methods. Food Control, Amsterdam, v. 16, p. 753-759, 2005. https://doi.org/10.1016/j.foodcont.2004.06.015 HEID, C.A.; STEVES, J.; LIVAK, K. J.; WILLIAMS, P. M. Real time quantitative PCR. Genome Research, New York, v. 6, n. 10, p. 986-994, 1996. https://doi.org/10.1101/gr.6.10.986 HOLST-JENSEN, A.; RONNING, S. B.; LOVSETH, A.; BERDAL, K. G. PCR technology for screening and quantification of genetically modified organisms (GMOs). Analytical and Bioanalytical Chemistry, Berlin, v. 375, p. 985-993, 2003. https://doi.org/10.1007/s00216-003-1767-7 LEE, S. H.; MIN, D. M., KIM, J. K. Qualitative and quantitative polymerase chain reaction analysis for genetically modified maize MON863. Journal of Agricultural and Food Chemistry, New York, v. 54, p. 1124-1129, 2006. https://doi.org/10.1021/jf052199a LI, X.; PAN, L.; LI, J.; ZHANG, Q.; ZHANG, S.; LV, R.; YANG, L. Establishment and application of event- specific polymerase chain reaction methods for two genetically modified soybean events, A2704-12 and A5547-127. Journal of Agricultural and Food Chemistry, New York, v. 59, p. 13188-13194, 2011. https://doi.org/10.1021/jf202806w MAFRA, I., Silva, S. A., Moreira, E. J. M. O., da Silva, C. S. F., Beatriz, M., Oliveira, P. P. Comparative study of DNA extraction methods for soybean derived food products. Food Control, Amsterdam, v. 19, p. 1183– 1190, 2008. https://doi.org/10.1016/j.foodcont.2008.01.004 MAGHARI, B. M.; ARDEKANI, A. M. Genetically modified foods and social concerns. Avicenna Journal of Medical Biotechnology, Tehran, v. 3, n. 3, p. 109-117, 2011. MATSUOKA, T.; KAWASHIMA, Y.; AKIYAMA, H.; MIURA, H.; GODA, Y.; SEBATA, T.; ISSHIKI, K.; TOYODA, M.; HINO, A. A detection method for recombinant DNA from genetically modified soybeans and processed foods containing them. Journal of the Food Hygienic Society of Japan, Tokyo, v. 40, p. 149-157, 1999. https://doi.org/10.3358/shokueishi.40.149 PINTO, G. B. A.; SILVA, M.; GREINER, R.; KONIETZNY, U.; SOCCOL, C. R.; SPIER, M. R.; FILHO, M. A. S. C.; PANDEY, A.; THOMAZ-SOCCOL, V. Application of Polymerase Chain Reaction for high sensitivity detection of roundup readyTM soybean seeds and grains in varietal mixtures. Food Technology and Biotechnology, Kačićeva, v. 49, n. 3, p. 277-285, 2011. ROTT, M. E.; LAWRENCE, T. S.; WALL, E. M.; GREE, M. J. Detection and quantification of roundup ready soy in foods by conventional and real-time polymerase chain reaction. Journal of Agricultural and Food Chemistry, New York, v. 52, p. 5223-5232, 2004. https://doi.org/10.1021/jf030803g SIEW-PING, K.; YOKE-KQUEEN, C.; SON, R. Quantitative analysis of Roundup Ready soybean content in soy-derived food and animal feed by using Real-time PCR incorporated with cloned DNA fragments. International Food Research Journal, Serdang, v. 18. p. 507-514, 2011. 41 Real-time PCR... LEÃO-BUCHIR, J. et al Biosci. J., Uberlândia, v. 34, n. 1, p. 34-41, Jan./Feb. 2018 SCHOLTENS, I. M. J.; KOK, E. J.; HOUGS, L.; MOLENAAR, B.; THISSEN, J. T. N. M.; VAN DER VOET, H. Increased efficacy for in-house validation of real-time PCR GMO detection methods. Analytical and Bioanalytical Chemistry, Berlin, v. 396, p. 2213- 2227, 2010. https://doi.org/10.1007/s00216-009-3315-6 SHIMIZU, E.; KATO, H.; NAKAGAWA, Y.; KODAMA, T.; FUTO, S.; MINEGISHI, Y.; WATANABE, T.; AKIYAMA, H.; TESHIMA, R.; FURUI, S.; HINO, A.; KITTA, K. Development of a screening method for genetically modified soybean by plasmid- based quantitative competitive polymerase chain reaction. Journal of Agricultural and Food Chemistry, New York, v. 56, p. 5521-5527, 2008. https://doi.org/10.1021/jf073348n TUNG NGUYEN, C. T.; SON, R.; RAHA, A. R.; LAI, O. M.; CLEMENTE MICHAEL WONG, V. L. Detection of genetically modified organisms (GMOs) using molecular techniques in food and feed samples from Malaysia and Vietnam. International Food Research Journal, Serdang, v. 15, n 2, p. 155-166, 2008. VILJOEN, C. D.; DAJEE, B. K.; BOTHA, G. M. Detection of GMO in food products in South Africa: Implications of GMO labelling. African Journal of Biotechnology, Nairóbi, v. 5, n 2, p. 73-82, 2006.