Single-laboratory validation of a method for detection of Roundup Ready soy in soybeans: application of new strategies for qualitative validation

Main Article Content

C.S.W. Miaw
G.C.S. Amâncio
V.N. Rocha
J.E.G.C. Madeira
S.V.C. Souza

Keywords

genetically modified organisms, screening method, single-laboratory validation, Roundup Ready soy, labelling regulations

Abstract



Considering expansion of genetically modified organisms and the basic principle of consumers’ right to access information about products, legislations of several countries establish a limit for labelling transgenic food. Qualitative tests based on the polymerase chain reaction (PCR) have been recommended. However, validation of qualitative methods is still a critical point in the quality management of food analysis laboratories. A screening method for the detection of Roundup Ready (RR) soy in soybeans by nested PCR was validated by a novel qualitative approach, in a single-laboratory, considering all fundamental parameters for an adequate evaluation of fitness for purpose. Blank samples of soybeans and formulations containing 0.001 to 1% of RR soy were analysed. Agarose gel electrophoresis and fluorimetry techniques were compared in terms of the quantification of extracted DNA. False-positive rate obtained was 0%, with selectivity and reliability rates of 100.0% for both techniques. Sensitivity and reliability rates varied between 23.3 and 100.0% (agarose gel) and between 30.0 and 100.0% (fluorimetry), respectively. Levels above 0.030% presented 100.0% positive results. Unreliable regions were estimated by non-linear models, and the calculated detection limits were 0.0067 and 0.0047%, for agarose gel electrophoresis and fluorimetry, respectively. Accordance and concordance values of 1.0 were obtained for levels near the regulated limit. The method was considered fit for screening purposes. Analysis of commercial samples demonstrated the applicability of the method and the compliance with Brazilian legislation.




 
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References

Bahrdt, C., Krech, A.B., Wurz, A. and Wulff, D., 2010. Validation of a newly developed hexaplex real-time PCR assay for screening for presence of GMOs in food, feed and seed. Analytical and Bioanalytical Chemistry 396: 2103-2112.
Brasil, 2003. Decret number 4.680. April 24, 2003. Union Official Diary, Brasília, DF, Brazil.
Bressani, R., 1975. Nutritional contribution of soy protein to food systems. Journal of the American Oil Chemists’ Society 52: 254-262.
Cárdenas, S. and Valcárcel, M., 2005. Analytical features in qualitative analysis. Trends in Analytical Chemistry 24: 477-487.
Codex Alimentarius, 2010. Alinorm 10/33/23. Joint FAO/WHO food standards programme Codex Alimentarius commission 33th session, Geneva – report of the thirty-first session of the Codex Committee on methods of analysis and sampling, Budapest, Hungary. Available at: http://tinyurl.com/9ys3qvn.
European Commission (EC), 2002. Commission decision 202/657/EC of 12 August 2002. Implementing Council Directive 96/23/EC concerning performance of analytical methods and the interpretation of results. Official Journal of the European Communities L221: 8.
European Network of GMO Laboratories (ENGL), 2015. Definition of minimum performance requirements for analytical methods of GMO testing. Available at: http://tinyurl.com/j4znvqk.
European Union (EU), 2003. Traceability and labeling of GMOs. Available at: http://tinyurl.com/z6utxj7.
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.
Hill, A.R.C. and Reynolds, S.L., 1999. Guidelines for in-house validation of analytical methods for pesticide residues in food and animal feeds. Analyst 124: 953-958.
International Standard Organization (ISO), 2005. ISO/IEC 17025. General requirements for the competence of testing and calibration laboratories. ISO, Geneva, Switzerland.
Joint Research Center (JRC), 2005. Event-specific method for the quantification of maize line NK603 using real-time PCR Protocol. Available at: http://tinyurl.com/jev2zye.
Joint Research Center (JRC), 2011. Reference reports. Compendium of reference methods for GMO analysis. Available at: http://tinyurl.com/zllfa2m.
Kodama, T., Kasahara, M., Minegishi, Y., Futo, S., Sawada, C., Watai, M., Akiyama, H., Teshima, R., Kurosawa, Y., Furui, S., Hino, A. and Kitta, K., 2011. Qualitative PCR method for Roundup Ready soybean: interlaboratory study. Journal of AOAC International 94: 224-231.
Leimanis, S., Hamels, S., Nazé, F., Sneyers, M., Mbella, G.M. and Hochegger, R., 2008. Validation of the performance of a GMO multiplex screening assay based on microarray detection. European Food Research and Technology 227: 1621-1632.
Lipp, M., Bluth, A., Eyquem, F., Kruse, L., Schimmel, H., Van den Eede, G. and Anklam, E., 2001. Validation of a method based on polymerase chain reaction for the detection of genetically modified organisms in various processed foodstuffs. European Food Research Technology 212: 497-502.
Lipp, M., Brodmann, P., Pietsch, K., Pauwels, J. and Anklam, E., 1999. IUPAC collaborative trial study of a method to detect genetically modified soy beans and maize in dried powder. Journal of AOAC International 82: 923-928.
Mano, J., Harada, M., Takabatake, R., Furui, S., Kitta, K., Nakamura, K., Akiyama, H., Teshima, R., Noritake, H., Hatano, S., Futo, S., Minegishi, Y. and IIzuka, T., 2012. Comprehensive GMO detection using real-time PCR array: single-laboratory validation. Journal of AOAC International 95: 508-516.
Magnusson, B. and Örnemark, U., 2014. Eurachem guide: the fitness for purpose of analytical methods – a laboratory guide to method validation and related topics. Available at: http://tinyurl.com/zmvga22.
Marcelino, F.C., Guimarães, M.F.M. and De-Barros, E.G., 2008. Detection and quantification of Roundup Ready® soybean residues in sausage samples by conventional and real-time PCR. Ciência e Tecnologia de Alimentos 28: 38-45.
Meyer, R. and Jaccaud, E., 1997. Detection of genetically modified in processed food production: development and validation of a PCR assay for the specific detection of glyphosate tolerant soybeans. In: Proceedings of the 9th European Conference on Food Chemistry, Interlaken, Switzerland, 1, pp. 23-28.
Pulido, A., Ruisánchez, I., Boqué, R. and Rius, F.X., 2003. Uncertainty of results in routine qualitative analysis. Trends in Analytical Chemistry 22: 647-654.
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, Joint Research Centre, World Health Organization. Available at: http://tinyurl.com/zw7rg5q.
Ríos, A., Barceló, D., Buydens, L., Cárdenas, S., Heydorn, K., Karlberg, B., Klemm, K., Lendl, B., Milman, B., Neidhart, B., Stephany, R.W., Townshend, A., Zschunke, A. and Valcárcel, M., 2003. Quality assurance of qualitative analysis in the framework of the European project ‘MEQUALAN’. Accreditation and Quality Assurance 8: 68-77.
Sieradzki, Z., Mazur, M. and Kwiatek, K., 2008. Validation of procedures based on PCR reactions for detection and identification of genetically modified maize and soybean. Bulletin of the Veterinary Institute in Pulawy 52: 611-614.
Taverniers, I., Windels, P., Van Bockstaele, E. and De Loose, M., 2001. Use of cloned DNA fragments for event-specific quantification of genetically modified organisms in pure and mixed food products. European Food Research and Technology 213: 417-424.
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), 2014. Production, supply and distribution online. Available at:http://www.fas.usda.gov/psdonline/psdQuery.aspx.
Van der Voet, H., Van Rhijn, J.A.H., Van de Wiel, H.J., 1999. Inter-laboratory, time, and fitness-for-purpose aspects of effective validation. Analytica Chimica Acta 391: 159-171.
Wood, R., 1999. How to validate analytical methods. Trends in Analytical Chemistry 18: 624-632.