Extraction of DNA from different sample types – a practical approach for GMO testing


  • Jana Žel
  • Tina Demšar
  • Dejan Štebih
  • Mojca Milavec
  • Kristina Gruden




Extraction methods, Genetically modified organisms (GMO), Decision-making system, GMO testing, NucleoSpin® Food, Cetyltrimethylammonium bromide (CTAB)


Current methods based on DNA targets for the detection, identification and quantification of genetically modified organisms (GMOs) involve extraction of the DNA. Different extraction procedures have been developed for the great variety of samples from food, feed, seeds and particular plant parts. This makes the operation of routine analytical laboratories complex and workloads heavy. Here we present a decision-making system, developed over many years of GMO testing on different samples, that result in the application of only a few extraction methods for the majority of samples. Developed decision-making system enables quicker and more cost effective testing of GMOs. In addition, the performance of DNA extraction resulting from the use of the selected extraction methods is presented for use in subsequent testing of GMOs by real time PCR methods. This approach can be used as a model for similar systems based on nucleic acid analysis in food, feed, seeds and plants.


Bellocchi, G., De Giacomo, M., Foti, N., Mazzara, M., Palmaccio, E., Savini, C., Di Domenicantonio, C., Onori, R., Van den Eede, G., 2010. Testing the interaction between analytical modules: an example with Roundup Ready soybean line GTS 40-3-2. BMC Biotechnol., 10:55-69. DOI: https://doi.org/10.1186/1472-6750-10-55

Bustin, S.A., Benes, V., Garson, J.A., Hellemans, J., Huggett, J., Kubista, M., Mueller, R., Nolan, T., Pfaffl, M.W., Shipley, G.L., Vandesompele, J., Wittwer, C.T., 2009. The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin. Chem., 55 (4), 611-22. DOI: https://doi.org/10.1373/clinchem.2008.112797

Cankar, K., Stebih, D., Dreo, T., Zel, J., 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 Biotechnol., 6:37-52.

Codex Committee On Methods Of Analysis And Sampling, 2010. Guidelines on performance criteria and validation of methods for detection, identification and quantification of specific DNA sequences and specific proteins in foods. CAC/GL 74-2010 Codex alimentarius commission WHO Rome.

Costa, J., Mafra, I., Amaral, J.S., Oliveira, M.B.P.P., 2010. Monitoring genetically modified soybean along the industrial soybean oil extraction and refining processes by polymerase chain reaction techniques. Food Res. Int., 43:301–306. DOI: https://doi.org/10.1016/j.foodres.2009.10.003

Demeke, T., Jenkins, G.R., 2010. Influence of DNA extraction methods, PCR inhibitors and quantification methods on real-time PCR assay of biotechnology-derived traits. Anal. Bioanal. Chem., 396:1977–1990. DOI: https://doi.org/10.1007/s00216-009-3150-9

European Network of GMO Laboratories, 2015. Definition of minimum performance requirements for analytical methods of GMO testing. JRC95544.

European Network of GMO laboratories (ENGL), 2011. Verification of analytical methods for GMO testing when implementing interlaboratory validated methods. European Commission; JRC Scientific and Technical Reports, EUR 24790 EN – 2011.

Genetic ID NA Inc., 2009. NOST-Spec Construct-specific Method for the Detection of CDC Triffid Flax (Event FP967) Using Real-time PCR. http://gmo-crl.jrc.ec.europa.eu/doc/Flax-CDCTriffidFlaxJRC091030.pdf

Guertler, P., Eicheldinger, A., Muschler, P., Goerlich, O., Busch, U., 2014. Automated DNA extraction from pollen in honey. Food Chem., 149:302–306. DOI: https://doi.org/10.1016/j.foodchem.2013.10.129

Gryson, N., 2010. Effect of food processing on plant DNA degradation and PCR-based GMO analysis: a review. Anal Bioanal. Chem., 396:2003–2022. DOI: https://doi.org/10.1007/s00216-009-3343-2

Hernandez, M., Duplan, M.N., Berthier, G., Vaitilingom, M., Hauser, W., Freyer, R., Pla, M., Bertheau, Y., 2004. Development and Comparison of Four Real-Time Polymerase Chain Reaction Systems for Specific Detection and Quantification of Zea mays L. J Agric. Food Chem., 52:4632–4637. DOI: https://doi.org/10.1021/jf049789d

Hernandez, M., Ferrando, A., Esteve, T., Puigdomenech, P., Prat, S., Pla, M., 2003. Real-time and conventional polymerase chain reaction systems based on the metallo-carboxypeptidase inhibitor gene for specific detection and quantification of potato and tomato in processed food. J Food Prot., 66:1063–107.0 DOI: https://doi.org/10.4315/0362-028X-66.6.1063

Holst-Jensen, A., Berdal, K., 2004. The modular analytical procedure and validation approach and the units of measurement for genetically modified materials in foods and feeds. J AOAC Int, 87:927–936. DOI: https://doi.org/10.1093/jaoac/87.4.927

ISO 21571: 2005 Foodstuffs - Methods of analysis for the detection of genetically modified organisms and derived products - Nucleic acid extraction. Geneva: International Organization for Standardization.

ISO 21571:2005/A1 2013 Foodstuffs - Methods of analysis for the detection of genetically modified organisms and derived products - Nucleic acid extraction - Amendment 1 (ISO 21571:2005/Amd1:2013) Geneva: International Organization for Standardization.

Lee, D., La Mura, M., Allnutt, T., Powell, W., Greenland, A., 2009. Isothermal amplification of genetically modified DNA sequences directly from plant tissues lowers the barriers to high-throughput and field-based genotyping. J Agric, Food Chem, 57:9400–9402. DOI: https://doi.org/10.1021/jf902455j

Mano, J., Hatano, S., Futo, S., Minegischi, Y., Ninomiya, K., Nakamura, K., Kondo, K., Teshima, R., Takabatakabe, R. and Kitta, K., 2014. Development of direct real-time PCR system applicable to a wide range of foods and agricultural products. Food Hyg, Saf, Sci, 55:25-33, DOI: https://doi.org/10.3358/shokueishi.55.25

Mazzara, M., Grazioli, E., Savini, C., Van den Eede, G., 2006. Validation Report and Protocol. Event-specific method for the quantitation of rice line llrice62 using real-time PCR validation report and Protocol. Institute for health and consumer protection. European Commission, JRC

Biotechnology and GMO unit. Milavec, M., Dobnik, D., Yang, L., Zhang, D., Gruden, K., Zel, J., 2014. GMO quantification: valuable

experience and insights for the future. Anal Bioanal Chem., 406:6485–6497.

Murray, M. G., Thompson, W. F., 1980. Rapid isolation of high molecular weight plant DNA. Nucleic Acids Research, 8:4321-4325. DOI: https://doi.org/10.1093/nar/8.19.4321

Pavšič, J., Žel, J., Milavec, M., 2015 Digital PCR for direct quantification of viruses without DNA extraction. Anal Bioanal Chem (DOI10.1007/s00216-015-9109-0, published on-line 19.October 2015) DOI: https://doi.org/10.1007/s00216-015-9109-0

Pauli, U., Linger, M., Schrott, M., Schouvey, B., Hubner, P., Brodmann, P., Eugster, A., 2001. Quantitative detection of genetically modified soybean and maize: Method evaluation in a swiss ring trial. Mitt Lebensm, Hyg., 92:145–158.

Pirondini, A., Bonas, U., Maestri, E., Visioli, G., Marmiroli, M., Marmiroli, N., 2010. Yield and amplificability of different DNA extraction procedures for traceability in the dairy food chain. Food Control, 21:663–668. DOI: https://doi.org/10.1016/j.foodcont.2009.10.004

Sacco, M.G., Gatto, F., Paracchini, V., Scaravelli, E., Nardini, E., Savini, C., Mazzara, M., Kreysa, J, 2014. Extraction of DNA from Choline Chloride Feed Additive ( CC ) and from derived and Screening of CC and PMCC for ( a ) presence of rice and ( b ) presence of BT63. European Commission JRC Science and Policy Reports. Smith, D.S., Maxwell, P.W., De Boer, S.H., 2005. Comparison of several methods for the extraction

of DNA from potatoes and potato-derived products. J Agric. Food Chem., 53:9848–9859.

Solfrizzo, M., Avantaggiato, G., Visconti, 1998. Use of various clean-up procedures for the analysis of ochratoxin A in cereals. J Chromatogr. A, 815:67–73. DOI: https://doi.org/10.1016/S0021-9673(98)00271-4

Terry, C. F., Harris, N., Parker, H.C. 2002. Detection of genetically modified crops and their derivatives: Critical steps in sample preparation and extraction. J AOAC Int., 85:768–774. DOI: https://doi.org/10.1093/jaoac/85.3.768

Waiblinger, H.U., Grohmann, L., 2014. Guidelines for validation of DNA extraction methods applied in subsequent PCR analysis of food and feed products for the presence of genetically modified material. J Verbr. Lebensm., 9:183–190. DOI: https://doi.org/10.1007/s00003-014-0862-3

Waiblinger, H.U., Ohmenhaeuser, M., Meissner, S., Schillinger, M., Pietsch, K., Goerlich, O., Mankertz, J., Lieske, K., Brol, H., 2012. In-house and interlaboratory validation of a method for the extraction of DNA from pollen in honey. J. Verbr. Lebensm., 7:243–254. DOI: https://doi.org/10.1007/s00003-012-0774-z

Zeitler, R., Pietsch, K., Waiblinger, H.U., 2002. Validation of real-time PCR methods for the quantification of transgenic contaminations in rape seed. Eur, Food Res. Technol., 214:346–351. DOI: https://doi.org/10.1007/s00217-001-0454-1

Žel, J., Milavec, M., Morisset, D., Plan, D., Van den Eede, G., Gruden, K., 2012. How to Reliably Test for GMOs. 1st ed. Springer, New York, 100pp. DOI: https://doi.org/10.1007/978-1-4614-1390-5






Original Research Paper

How to Cite

Žel, J., Demšar, T., Štebih, D., Milavec, M., & Gruden, K. (2015). Extraction of DNA from different sample types – a practical approach for GMO testing. Acta Biologica Slovenica, 58(2), 61-75. https://doi.org/10.14720/abs.58.2.15611

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