Standardization of PCR and sequencing-based methods for the detection of the presence of CP4 EPSPS gene in Zea mays
DOI:
https://doi.org/10.35699/2447-6218.2020.19990Keywords:
Genetically modified organisms, MON88017, Detection, MaizeAbstract
With the rise in planting of transgenic cultivars in Brazil as well as worldwide, the demand for legal regulations has increased. The transgenic event MON88017 is often found in maize cultivars marketed in Brazil. The event contains the CP4 EPSPS and cry3Bb1 genes, which encode tolerance to the herbicide glyphosate and resistance to caterpillars, respectively. Globally, methodologies to track transgenic events are mandatory. The objective of this study was to standardize a method based on qualitative PCR and sequencing for detection of the CP4 EPSPS gene in Zea mays. DNA was extracted from three commercial strains of transgenic maize, containing the MON88017 event, as well as conventional maize. Primers were designed for partial detection of the zein and CP4 EPSPS genes. PCR reactions were performed for detection of partial regions of CP4 EPSPS and Zein (as endogenous marker of Z. mays) genes. The three transgenic maize inbred lines tested positive for zein and CP4 EPSPS, and the two conventional strains tested negative for CP4 EPSPS and positive for the Zein gene. To confirm the presence of the genic regions, PCR products were sequenced and showed 100% identity with sequences of Zein and CP4 EPSPS genes deposited in GenBank. Thus, the results of this study suggest the applicability of an ‘in-house’ method for the qualitative detection of CP4 EPSPS in genetically modified maize cultivars.
References
Abdel-Latif, A.; G. Osman. 2017. Comparison of three genomic DNA extraction methods to obtain high DNA quality from maize. Plant Methods, 13: 1–9. Doi: https://doi.org/10.1186/s13007-016-0152-4.
Anklam, E.; Gadani, F.; Heinze, P.; Pijnenburg, H.; Den Van Eede, G. 2002. Analytical methods for detection and determination of genetically modified organisms in agricultural crops and plant-derived food products. European Food Research and Technology, 214: 3–26. Doi: https://doi.org/10.1007/s002170100415.
Brasil – CIB - Conselho de informações sobre biotecnologia. 2010. MON88017 (YieldGard VT Rootworm/RR2). Retrieved on April 04, 2019 from: https://www.isaaa.org/gmapprovaldatabase/event/default.asp?EventID=94
Brasil – MAPA - Ministério da Agricultura, Pecuária e Abastecimento. 2003. Decreto No. 4.680/2003: 1–2.
Broeders, S. R. M.; De Keersmaecker, S. C. J.; Roosens, N. H. C. 2012. How to Deal with the Upcoming Challenges in GMO Detection in Food and Feed. Journal of Biomedicine and Biotechnology, 2012: 1-11. Doi: https://doi.org/10.1155/2012/402418
Campos, L. F. L.; Silva, A. N. M.; David,M. E. D. dos Santos, J.; Cangussu, C. H. C.; Xavier, M. A. S.; Xavier, A. R. E. O. 2018. Eficiência de métodos de extração de DNA de tecido foliar de espécies transgênicas de Glycine max e Zea mays. Retrieved on April, 03, 2019. From: http://www.fepeg2018.unimontes.br/anais/download/3324f215-d852-4a8c-8992-b47182cce712
Cardarelli, P.; Branquinho, M. R.; Ferreira, R. T. B.; da Cruz, F. P.; Gemal, A. L. 2005. Detection of GMO in food products in Brazil: the INCQS experience. Food Control, 16: 859–866. Doi: https://doi.org/10.1016/j.foodcont.2004.07.010.
Cardoso, L.; Cangussu, C. H. C.; Nogueira, H. S.; dos Santos, J.; Xavier, M. A. S.; de Oliveira, A. M. E.; Albuquerque, C. J. B.; Xavier, A. R. E. O. 2018. Métodos baseados na detecção de DNA para rastreamento de modificações genéticas em cultivares transgênicos de milho e soja. Caderno de Ciências Agrárias, 9: 101–114. [online]. Acesso em 24/04/2020. Disponível em: https://pdfs.semanticscholar.org/7e54/34bb0f28066273f5aaf8e37a27f7ba3efbe3.pdf.
Conceição, F. R.; Moreira, A. N.; Binsfeld, P. C. 2006. Detecção e quantificação de organismos geneticamente modificados em alimentos e ingredientes alimentares. Ciência Rural, 36: 315-324. Doi: https://doi.org/10.1590/S0103-84782006000100053.
Cruz, J. C.; Pereira-Filho, I. A.; Borghi, E.; Simão, E. P. 2015. Quatrocentas e setenta e sete cultivares de milho estão disponíveis no mercado de sementes do Brasil para a Safra 2015/16. Embrapa Milho e Sorgo, 1: 1–29. [online]. Acesso em 24/04/2020. Disponível em: https://www.infoteca.cnptia.embrapa.br/infoteca/bitstream/doc/1038512/1/doc184.pdf
Dinon, A. Z.; Prins, T. W.; Van Dijk, J. P.; Arisi, A. C. M.; Scholten, I. M. J.; Kok, E. J. 2011. Development and validation of real-time PCR screening methods for detection of cry1A.105 and cry2Ab2 genes in genetically modified organisms. Analytical and Bioanalytical Chemistry, 400: 1433–1442. Doi: https://doi.org/10.1007/s00216-011-4875-9.
Doyle, J. J.; Doyle, J. L. 1990. Isolation of plant DNA from fresh tissue. Focus. 12: 13-15.
Forte, V. T.; Di Pinto, A.; Martino, C.; Tantillo, G. M.; Grasso, G.; Schena, F. P. 2005. A general multiplex-PCR assay for the general detection of genetically modified soya and maize. Food Control, 16: 535–539. Doi: https://doi.org/10.1016/j.foodcont.2004.05.010.
Fraiture, M. A.; Herman, P.; Taverniers, I.; De Loose, M.; Deforce, D.; Roosens, N. H. 2015. Current and new approaches in GMO Detection: challenges and solutions. Biomed Research International, 2015: 1–22. Doi: http://dx.doi.org/10.1155/2015/392872.
Gašparič, M. B.; Tengs, T.; La Paz, J. L.; Holst-Jensen, A.; Pla, M.; Esteve, T; Zel, J.; Gruden, K. 2010. Comparison of nine different real-time PCR chemistries for qualitative and quantitative applications in GMO detection. Analytical and Bioanalytical Chemistry, 396: 2023–2029. Doi: https://doi.org/10.1007/s00216-009-3418-0.
Germini, A.; Zanetti, A.; Salati, C.; Rossi, S.; Forre, C.; S. Schmid, S.; Fogher, C.; Marchelli, R. 2004. Development of a seven-target multiplex PCR for the simultaneous detection of transgenic soybean and maize in feeds and foods. Journal of Agricultural and Food Chemistry, 52: 3275–3280. Doi: https://doi.org/10.1021/jf035052x.
Heck, G. R.; Armstrong, C. L.; Astwood, J. D.; Behr, C. F.; Bookout, J. T.; Brown, S. M.; You, J. 2005. Development and characterization of a CP4 EPSPS-based, glyphosate-tolerant corn event. Crop Science, 45: 329-339. Doi: https://doi.org/10.2135/cropsci2005.0329.
Heide, B. R.; Heir, E.; Holck. A. 2008. Detection of eight GMO maize events by qualitative, multiplex PCR and fluorescence capillary gel electrophoresis. European Food Resesarch and Technology, 227: 527-535. Doi: https://doi.org/10.1007/s00217-007-0751-4.
Hernández, M.; Rodríguez-Lázaro, D.; Esteve, T.; Prat, S.; Pla, M. 2003. Development of melting temperature-based SYBR™Green I polymerase chain reaction methods for multiplex genetically modified organism detection. Analytical Biochemistry, 323: 164–170. Doi: https://doi.org/10.1016/j.ab.2003.07.015.
Höhne, M.; Santisi, C. R.; Meyer, R. 2002. Real-time multiplex PCR: an accurate method for the detection and quantification of 35S-CaMV promoter in genetically modified maize containing food. European Food Research and Technology, 215: 59-64. Doi: https://doi.org/10.1007/s00217-002-0503-4
Holst-Jensen, A., S. B. Rønning, A. Løvseth and K. G. Berdal. 2003. PCR technology for screening and quantification of genetically modified organisms (GMOs). Analyticaland Bioanalytical Chemistry 375: 985–93. [online]. Acesso em 24/04/2020. Disponível em: https://link.springer.com/content/pdf/10.1007/s00216-003-1767-7.pdf
Holst-Jensen, A., Y. Bertheau, M. de Loose, L. Grohmann, S. Hamels, L. Hougs, D. Morisset, S. Pecoraro, M. Pla, M. Van den Bulcke and D. Wulff. 2012. Detecting un-authorized genetically modified organisms (GMOs) and derived materials. Biotechnology Advances, 30: 1318–35. Doi: https://doi.org/10.1016/j.biotechadv.2012.01.024
ISAAA, 2018. Event Name: MON88017. Retrieved on April, 04, 2019. From: <http://www.isaaa.org/gmapprovaldatabase/event/default.asp?EventID=94>.
James, D.; Schmidt, A. M.; Wall, E.; Green, M.; Masri, S. 2003. Reliable detection and identification of genetically modified maize, soybean and canola by multiplex PCR analysis. Journal of Agricultural and Food Chemistry, 51: 5839–5834. Doi: https://doi.org/10.1021/jf0341159
Kim, J. H.; Song, H. S.; Heo, M. S.; Lee, W. Y.; Lee, S. H.; Park, S. H.; Park, H. K.; Kim, M. C.; Kim, H. Y. 2006. Detection of eight different events of genetically modified maize by multiplex PCR method. Food Science and Biotechnology, 15: 148–151. [online]. Acesso em 24/04/2020. Disponível em: http://www.koreascience.or.kr/article/JAKO200609905801382.page.
König, A.; Cockburn, A.; Crevel, R. W. R.; Debruyne, E.; Grafstroem, R.; Hammerling, U.; Kimber, I.; Knudsen, I.; Kuiper, H. A.; Peijnenburg, A. A. C. M.; Penninks, A. H.; Poulsen, M.; Schauzu, M.; Wal, J. M. 2004. Assessment of the safety of foods derived from genetically modified (GM) crops. Food and Chemical Toxicology, 42:1047–1088. Doi: https://doi.org/10.1016/j.fct.2004.02.019.
Marmiroli, N.; Maestri, E.; Gullì, M.; Malcevschi, A.; Peano, C.; Bordoni, R.; De Bellis. G. 2008. Methods for detection of GMOs in food and feed. Analytical and Bioanalytical Chemistry, 392: 369–384. Doi: https://doi.org/10.1007/s00216-008-2303-6.
Matsuoka, T.; Kuribara, H.; Takubo, K.; Akiyama, H.; Miura, H.; Goda, Y.; Kusakabe, Y.; Isshiki, K.; Toyoda, M.; Hino, A. 2002. Detection of recombinant DNA segments introduced to genetically modified maize (Zea mays). Journal of Agricultural and Food Chemistry, 50:2100-2109. Doi: https://doi.org/10.1021/jf011157t.
Mazza, M. C. M.; Bittencourt, J. V. M. 2000. Extração de DNA de tecido vegetal de Araucaria angustifolia (Araucariaceae). Boletim de Pesquisas Florestais, 41:12-17. Disponível em: https://www.alice.cnptia.embrapa.br/bitstream/doc/280588/1/mazza.pdf
Meyer, R. 1999. Development and application of DNA analytical methods for the detection of GMOs in food. Food Control, 10:391–399. Doi: https://doi.org/10.1016/S0956-7135(99)00081-X.
Miaw, C. S. W.; Amâncio, G.C.S.; Madeira, J. E. G. C.; Souza, S. V. C. 2014. Métodos para detecção de soja Roundup Ready® em grãos e produtos de soja por reação em cadeia da polimerase: revisão e análise crítica das práticas de validação. Revista do Instituto Adolfo Lutz, 73:9–25. Doi: https://doi.org/10.18241/0073-98552014731585.
Michelini, E.; Simoni, P.; Cevenini, L.; Mezzanotte, L.; Roda, A. 2008. New trends in bioinalitical tools for the detection of genetically modified organisms: an update. Analytical and Bioanalytical Chemistry, 392: 355–367. Doi: https://doi.org/10.1007/s00216-008-2193-7.
Milavec, M.; Dobnik, D.; Yang, L.; Zhang, D.; Gruden, K.; Žel, J. 2014. GMO quantification: valuable experience and insights for the future. Analytical and Bioanalytical Chemistry, 406: 6485–6497. Doi: https://doi.org/10.1007/s00216-014-8077-0.
Nascimento, V. E.; Pinho, E. V. R. V.; Pinho, R. G. V.; Santos, B. C.; Pereira, E. M.; Júnior, A. D. N. 2010. Especificidade e sensibilidade da técnica de PCR para detecção de milho geneticamente modificado. XXVIII Congresso Nacional de Milho e Sorgo – Goiânia: Associação Brasileira de Milho e Sorgo. CD-rom. Retrieved on September 15, 2019. From: http://abms.org.br/eventos_anteriores/cnms2010/trabalhos/0361.pdf.
Nikolić, Z.; Milošević, M.; Vujaković, M.; Marinković, D.; Jevtić, A.; Balešević-Tubić, S. 2008. Qualitative triplex PCR for the detection of genetically modified soybean and maize. Biotechnology & Biotechnological Equipment, 22: 801–803. Doi: https://doi.org/10.1080/13102818.2008.10817556.
Onishi, M.; Matsuoka, T.; Kodama, T.; Kashiwaba, K.; Futo, S.; Akiyama, H.; Maitani, T.; Furui, S.; Oguchi, T. Hino, A. 2005. Development of a multiplex polymerase chain reaction method for simultaneous detection of eight events of genetically modified maize. Journal of Agricultural and Food Chemistry, 53: 9713–9721. https://doi.org/10.1021/jf0515476.
Pereira-Filho, I. A.; Borghi, E. 2016. Mercado de sementes de milho no Brasil: safra 2016/2017. Embrapa Milho E Sorgo, 202: 1–28. [online]. Acesso em 24/04/2020. Disponível em: https://www.infoteca.cnptia.embrapa.br/infoteca/bitstream/doc/1060346/1/doc2021.pdf
Schmutz, J., Cannon, S. B.; Schlueter, J.; Ma, J.; Mitros, T.; Nelson, W.; Hyten, D. L.; Song, Q.; Thelen, J. J.; Cheng, J.; Xu, D.; Hellsten, U.; May, G. D.; Yu, Y.; Sakurai, T.; Umezawa, T.; Bhattacharyya, M. K.; Sandhu, D.; Valliyodan, B.; Lindquist, E.; Peto, M.; Grant, D.; Shu, S.; Goodstein, D.; Barry, K.; Futrell-Griggs, M.; Abernathy, B.; Du, J.; Tian, Z.; Zhu, L.; Gill, N.; Joshi, T.; Libault, M.; Sethuraman, A.; Zhang, X. C.; Shinozaki, K.; Nguyen, H. T.; Wing, R. A.; Cregan, P.; Specht, J.; Grimwood, J.; Rokhsar, D.; Stacey, G.; Shoemaker, R. C.; Jackson, S. A. 2010. Genome sequence of the palaeopolyploid soybean. Nature, 463: 178–183. Doi: https://doi.org/10.1038/nature08670.
Sika, C. K.; Kefela, T.; Adoukonou-Sagbadja, H.; Ahoton, L.; Saidou, A.; Baba-Moussa, L.; Baptiste, L. J.; Kotconi, S. O.; Gachomo, E. W. 2015. A simple and efficient genomic DNA extraction protocol for large scale genetic analyses of plant biological systems. Plant Gene, 1: 43–45. Doi: https://doi.org/10.1016/j.plgene.2015.03.001.
Tabima-Cubillos, L. Y.; Chaparro-Giraldo, A. Trujillo-Güiza, M. L. 2016. Detección de proteínas transgénicas en harinas de maíz comercializadas en Bogotá, Colômbia. Revista de Salud Publica, 18: 470-483. Doi: http://dx.doi.org/10.15446/rsap.v18n3.46923.
Tran, L. S P.; Mochida, K. 2010. Functional genomics of soybean for improvement of productivity in adverse conditions. Functional and Integrative Genomics, 10: 447–462. Doi: https://doi.org/10.1007/s10142-010-0178-z.
Vidal, N.; Barbosa, H.; Jacob, S.; Arruda, M. 2015. Comparative study of transgenic and non-transgenic maize (Zea mays) flours commercialized in Brazil, focussing on proteomic analyses. Food Chemistry, 180: 288–294. Doi: https://doi.org/10.1016/j.foodchem.2015.02.051.
Vieira, F. A.; Santana, J. A. S.; Santos, R. M.; Fajardo, G. C.; Coelho, G. A. O.; Carvalho, D. 2010. Métodos de extração de DNA e seleção de primers de cpDNA para Ficus bonijesulapensis (Moraceae). Revista Caatinga, 23: 69–74. [online]. Acesso em 24/04/2020. Disponível em: https://periodicos.ufersa.edu.br/index.php/caatinga/article/view/1403
Wu, H.; Zhang, Y.; Zhu, C.; Xiao, X.; Zhou, X.; Xu, S.; Shen, W.; Huang, M. 2012. Presence of CP4-EPSPS Component in Roundup Ready Soybean-Derived Food Products. International Journal of Molecular Science, 13: 1919–1932. Doi: https://doi.org/10.3390/ijms13021919.
Yamaguchi, H.; Sasaki, K.; Umetsu, H. Kamada, H. 2003. Two detection methods of genetically modified maize and the state of its import into Japan. Food Control, 14: 201- 206. Doi: https://doi.org/10.1016/S0956-7135(02)00062-2.
Zhang, D.; Guo, J. 2011. The development and standardization of testing methods for genetically modified organisms and their derived products. Journal of Integrative Plant Biology, 53: 539–551. Doi: https://doi.org/10.1111/j.1744-7909.2011.01060.x.
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