Bt11 event detection by real-time PCR: single-laboratory validation, comparison of DNA extraction and quantification techniques and application

Main Article Content

C.S.W. Miaw
E.N. Vasconcelos
N.C.C. Guimarães
S.V.C. Souza

Keywords

qualitative method validation, genetically modified organisms, Bt11 maize, real-time PCR, DNA extraction, DNA quantification

Abstract

The detection of genetically modified organisms (GMO) by real-time polymerase chain reaction (PCR) is recommended due to its effectiveness in GMO analysis. A complete in-house validation method was applied to the detection of Bt11 events by real-time PCR. A full factorial design was used to compare DNA extraction (cetyltrimethyl ammonium bromide; CTAB, and NucleoSpin® Plant II Kit) and DNA quantification techniques (conventional GENESYS™ 10S UV-Vis spectrophotometer and confined drop-based NANOVUE™ Plus spectrophotometer). In the validation, various levels (0.0007 to 0.0315%) of Bt11 maize were formulated with blank maize and certified Bt11 reference material. A false-positive rate of 0% was obtained for blank samples, which corresponded to selectivity and reliability rates of 100%. The false-negative rate varied from 0 to 83.3%, consistent with sensitivity and reliability rates ranging from 16.7 to 100%. The Bt11 level that presented 100% positive results was 0.0315%, which indicated the sensitivity of the method. Non-linear models were used to estimate the region of unreliability and to calculate the detection limit of 0.014%. Accordance and concordance values of 1.0 were obtained for the 0.0315% level, which indicated method standardisation. Selectivity in the presence of interference was confirmed by the detection of Bt11 maize in the presence of other events. The method was considered robust for different DNA extraction and DNA quantification techniques. Higher DNA concentration values were obtained using CTAB. The absorbance ratio of A260/A230 was negatively influenced by quantification using a conventional spectrophotometer. Both DNA extraction techniques gave values of A260/A280 higher than 1.7, which indicated DNA of great purity. This validated method was applied to routine samples.

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References

Anvisa, 2003. Decree no. 4680, 24th April, 2003. Anvisa, Brasília, Brazil. Available at: http://portal.anvisa.gov.br/.
Cankar, K., Stebih, D., Dreo, T., Zel, J. and Gruden, K., 2006. Critical points of DNA quantification by real-time PCR – Effects of DNA extraction method and sample matrix on quantification of genetically modified organisms. BMC Biotechnology 6: 37.
Cárdenas, S. and Valcárcel, M., 2005. Analytical features in qualitative analysis. Trends in Analytical Chemistry 24(6): 477-487.
Celeres, 2016. 2° levantamento de adoção da biotecnologia agrícola no Brasil, safra 2015/16. Celeres, Uberlândia, Brazil. Available at: http://tinyurl.com/h559agh.
Codex Alimentarius, 2010. Alinorm 10/33/23. 2010. Joint FAO/WHO food standards programme Codex Alimentarius commission thirty-third session, Genebra – Report of the thirty-first session of the Codex Committee on methods of analysis and sampling, Budapeste. Codex Alimentarius, Rome, Italy. Available at: http://tinyurl.com/9ys3qvn.
Comissão Técnica Nacional de Biossegurança (CTNBio), 2008. Technical opinion no. 1255/2008 – Commercial release of genetically modified corn, Bt11 Corn. CTNBio, Brasilia, Brazil. Available at: http://tinyurl.com/zq7lkxj.
Comissão Técnica Nacional de Biossegurança (CTNBio), 2014. Commercial approval. CTNBio, Brasilia, Brazil. Available at: http://www.ctnbio.gov.br/.
Companhia Nacional de Abastecimento (CONAB), 2016. Acompanhamento da safra brasileira de grãos. v. 4, Safra 2015/16. CONAB, Brasilia, Brazil, pp. 1-154.
Duarte, J., 2000. Cultivo do Milho: importância econômica. Sete Lagoas: EMBRAPA Milho e Sorgo. Available at: http://tinyurl.com/grhq889.
Ellison, S.L.R. and Fearn, T., 2005. Characterising the performance of qualitative analytical methods: statistics and terminology. Trends in Analytical Chemistry 24: 468-476.
European Network of GMO Laboratories (ENGL), 2015. Definition of minimum performance requirements for analytical methods of GMO testing. Joint Research Centre, Ispra, Italy, 24 pp. Available at: http://tinyurl.com/j4znvqk.
European Union (EU), 2003. Traceability and labeling of GMOs. EU, Brussels, Belgium. Available at: http://tinyurl.com/z6utxj7.
Food and Agriculture Organization of the United Nations (FAO), 1983. Changing patterns and trends in feed utilization. FAO Economic and Social Development Paper, FAO, Rome, Italy, 65 pp.
GE Healthcare, 2010. NanoVue™ Plus spectrophotometer. GE Healthcare UK Limited, Little Chalfont, UK. Available at: https://ca.vwr.com/assetsvc/asset/en_CA/id/7120651/contents.
Germini, A., Zanetti, A., Salati, C., Rossi, S., Forré, C., Schmid, S. and Marchelli, R, 2004. Development of 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.
Gondim, C.S., Coelho, O.A.M., Alvarenga, R.L., Junqueira, R.G. and Souza, S.V.C., 2014. An appropriate and systematized procedure for validating qualitative methods: its application in the detection of sulfonamide residues in raw milk. Analytica Chimica Acta 830: 11-22.
Gruère, G.P. and Rao, S.R., 2007. A review of international labeling policies of genetically modified food to evaluate India’s proposed rule. AgBioForum 10: 51-66.
Hamels, S., Glouden, T., Gillard, K., Mazzara, M., Debode, F., Foti, N., Sneyers, M., Nuez, T.E., Pla, M., Berben, G., Moens, W., Bertheau, Y., Audéon, C., Van den Ede, G. and Remacle, J., 2009. A PCR-microarray method for the screening of genetically modified organisms. European Food Research and Technology 228: 531-541.
International Standard Organization (ISO), 2005a. ISO 21570. Foodstuffs – Methods of analysis for the detection of genetically modified organisms and derived products. Quantitative nucleic acid based methods. ISO, Geneva, Switzerland.
International Standard Organization (ISO), 2005b. ISO 21571. Foodstuffs – Methods of analysis for the detection of genetically modified organisms and derived products. Nucleic acid extraction. ISO, Geneva, Switzerland.
James, C., 2014. Global status of commercialized biotech/GM crops: 2013. Executive summary. ISAAA brief Ithaca, NY, USA, 49 pp.
Joint Research Center (JRC), 2011. Compendium of reference methods for GMO analysis. JRC, Brussels, Belgium. Available at: http://tinyurl.com/jrwbcuc.
Kheyrodin, H. and Ghazvinian, K., 2012. DNA purification and isolation of genomic DNA from bacterial species by plasmid purification system. African Journal of Agricultural Research 7: 433-442.
Kim, J.H., Zhang, D. and Kim, H.Y., 2014. Detection of sixteen genetically modified maize events in processed foods using four event-specific pentaplex PCR systems. Food Control 35: 345-353.
Macherey-Nagel, 2014. Genomic DNA from plant. User manual. Available at: http://tinyurl.com/zemejny.
Marmiroli, N., Maestri, E., Gullì, M., Malcevschi, A., Peano, C., Bordoni, R. and De Bellis, G., 2008. Methods for detection of GMOs in food and feed. Analytical and Bioanalytical Chemistry 392: 369-384.
Nuss, E.T. and Tanumihardjo, S.A., 2010. Maize: a paramount staple crop in the context of global nutrition. Comprehensive Reviews in Food Science and Food Safety 9: 417-436.
Pena-Pereira, F., Costas-Mora, I., Romero, V., Lavilla, I. and Bendicho, C., 2011. Advances in miniaturized UV-Vis spectrometric systems. Trends in Analytical Chemistry 30: 1637-1648.
Querci, M., Jermini, G. and Van den Eede, G., 2006. Training course on the analysis of food samples for the presence of genetically modified organisms: user manual. European Commission, Brussels, Belgium, 239 pp.
Ranum, P., Peña-Rosas, J.P. and Garcia-Casal, M.N., 2014. Global maize production, utilization, and consumption. Annals of New York Academy of Sciences 1312: 105-112.
Snedecor, G.W. and Cochran, W.G., 1989. Statistical methods. Iowa State University, Ames, IA, USA, pp. 149-176.
Thomson, J., 2003. Genetically modified food crops for improving agricultural practice and their effects on human health. Trends in Food Science and Technology 14: 210-228.
United Stated Department of Agriculture (USDA), 2016. Production, supply and distribution online. USDA, Washington, DC, USA. Available at: http://tinyurl.com/zfonm5s.
Xin, Z. and Chen, J., 2012. A high throughput DNA extraction method with high yield and quality. Plant Methods 8: 26.