Share This Article:

Application of Digital PCR in the Analysis of Transgenic Soybean Plants

Full-Text HTML XML Download Download as PDF (Size:478KB) PP. 403-417
DOI: 10.4236/abb.2016.710039    829 Downloads   1,187 Views  


Detection and quantification of transgenes are important in analyzing genetically modified organisms (GMOs). Quantitative polymerase chain reaction (qPCR) is commonly utilized for such purposes. However, qPCR has certain limitations in detecting and quantifying transgenes in GMOs, such as the need of certified reference materials, a standard curve, and possible affection by inhibitors. Therefore, alternative and possibly better methods are needed. Recent advances in digital PCR technologies have promised to allow accurate quantification of nucleic acids and therefore provided another useful technique to analyze GMOs. Thermo Fisher ScientificTM has recently commercialized the Applied BiosystemsTM QuantStudioTM 3D digital PCR system that can be used for a wide range of applications involving nucleic acids. It will be beneficial to the scientific community to show the applicability of this digital PCR system in detecting and quantifying transgenes in GMOs. In the present study, the transgenes present in the Roundup Ready Soybean (RR1, event 40-3-2) and Roundup Ready Soybean 2 (RR2, event MON89788) developed by Monsanto Corporation were analyzed by using this digital PCR system. The qPCR analysis results were included for comparison. Using specifically designed TaqMan assays, as low as 1% of the RR1 or RR2 soybean material was reliably detected and quantified on the dPCR platform. Therefore, digital PCR is a sensitive and reliable method to analyze the RR transgenic soybeans, and should be another useful tool for analyzing other transgenic plants.

Cite this paper

Wan, J. , Song, L. , Wu, Y. , Brzoska, P. , Keys, D. , Chen, C. , Valliyodan, B. , Shannon, J. and Nguyen, H. (2016) Application of Digital PCR in the Analysis of Transgenic Soybean Plants. Advances in Bioscience and Biotechnology, 7, 403-417. doi: 10.4236/abb.2016.710039.


[1] Paparini, A. and Romano-Spica, V. (2004) Public Health Issues Related with the Consumption of Food Obtained from Genetically Modified Organisms. Biotechnology Annual Review, 10, 85-122.
[2] Varzakas, T.H., Arvanitoyannis, I.S. and Baltas, H. (2007) The Politics and Science behind GMO Acceptance. Critical Reviews in Food Science and Nutrition, 47, 335-361.
[3] Hug, K. (2008) Genetically Modified Organisms: Do the Benefits Outweigh the Risks? Medicina (Kaunas), 44, 87-99.
[4] Rajan, S.R. and Letourneau, D.K. (2012) What Risk Assessments of Genetically Modified Organisms Can Learn from Institutional Analyses of Public Health Risks. Journal of Biomedicine and Biotechnology, 2012, Article ID: 203093.
[5] Kamle, S. and Ali, S. (2013) Genetically Modified Crops: Detection Strategies and Biosafety Issues. Gene, 522, 123-132.
[6] Lucht, J.M. (2015) Public Acceptance of Plant Biotechnology and GM Crops. Viruses, 7, 4254-4281.
[7] Ahmed, F.E. (2002) Detection of Genetically Modified Organisms in Foods. Trends in Biotechnology, 20, 215-223.
[8] Elenis, D.S., Kalogianni, D.P., Glynou, K., Ioannou, P.C. and Christopoulos, T.K. (2008) Advances in Molecular Techniques for the Detection and Quantification of Genetically Modified Organisms. Analytical and Bioanalytical Chemistry, 392, 347-354.
[9] Michelini, E., Simoni, P., Cevenini, L., Mezzanotte, L. and Roda, A. (2008) New Trends in Bioanalytical Tools for the Detection of Genetically Modified Organisms: An Update. Analytical and Bioanalytical Chemistry, 392, 355-367.
[10] Holst-Jensen, A. (2009) Testing for Genetically Modified Organisms (GMOs): Past, Present and Future Perspectives. Biotechnology Advances, 27, 1071-1082.
[11] Querci, M., Van den Bulcke, M., Zel, J., Van den Eede, G. and Broll, H. (2010) New Approaches in GMO Detection. Analytical and Bioanalytical Chemistry, 396, 1991-2002.
[12] Broeders, S.R., De Keersmaecker, S.C. and Roosens, N.H. (2012) How to Deal with the Upcoming Challenges in GMO Detection in Food and Feed. Journal of Biomedicine and Biotechnology, 2012, Article ID: 402418.
[13] Holst-Jensen, A., Bertheau, Y., de Loose, M., Grohmann, L., Hamels, S., Hougs, L., et al. (2012) Detecting Unauthorized Genetically Modified Organisms (GMOs) and Derived Materials. Biotechnology Advances, 30, 1318-1335.
[14] Milavec, M., Dobnik, D., Yang, L., Zhang, D., Gruden, K., and Zel, J. (2014) GMO Quantification: Valuable Experience and Insights for the Future. Analytical and Bioanalytical Chemistry, 406, 6485-6497.
[15] Fraiture, M.A., Herman, P., Taverniers, I., De Loose, M., Deforce, D. and Roosens, N.H. (2015) Current and New Approaches in GMO Detection: Challenges and Solutions. BioMed Research International, 2015, Article ID: 392872.
[16] Salihah, N.T., Hossain, M.M., Lubis, H. and Ahmed, M.U. (2016) Trends and Advances in Food Analysis by Real-Time Polymerase Chain Reaction. Journal of Food Science and Technology, 53, 2196-1209.
[17] Dobnik, D., Spilsberg, B., Bogozalec Kosir, A., Holst-Jensen, A. and Zel, J. (2015) Multiplex Quantification of 12 European Union Authorized Genetically Modified Maize Lines with Droplet Digital Polymerase Chain Reaction. Analytical Chemistry, 87, 8218-8226.
[18] Fu, W., Zhu, P., Wang, C., Huang, K., Du, Z., Tian, W., et al. (2015) A Highly Sensitive and Specific Method for the Screening Detection of Genetically Modified Organisms Based on Digital PCR without Pretreatment. Scientific Reports, 5, 12715.
[19] Holst-Jensen, A., Spilsberg, B., Arulandhu, A.J., Kok, E., Shi, J. and Zel, J. (2016) Application of Whole Genome Shotgun Sequencing for Detection and Characterization of Genetically Modified Organisms and Derived Products. Analytical and Bioanalytical Chemistry, 408, 4595-4614.
[20] Gerdes, L., Iwobi, A., Busch, U. and Pecoraro, S. (2016) Optimization of Digital Droplet Polymerase Chain Reaction for Quantification of Genetically Modified Organisms. Biomolecular Detection and Quantification, 7, 9-20.
[21] Zhu, P., Wang, C., Huang, K., Luo, Y. and Xu, W. (2016) A Novel Pretreatment-Free Duplex Chamber Digital PCR Detection System for the Absolute Quantitation of GMO Samples. International Journal of Molecular Sciences, 17, 402.
[22] Sykes, P.J., Neoh, S.H., Brisco, M.J., Hughes, E., Condon, J. and Morley, A.A. (1992) Quantitation of Targets for PCR by Use of Limiting Dilution. Biotechniques, 13, 444-449.
[23] Vogelstein, B. and Kinzler, K.W. (1999) Digital PCR. Proceedings of the National Academy of Sciences? of the United States of America, 96, 9236-9241.
[24] Bhat, S., Herrmann, J., Armishaw, P., Corbisier, P. and Emslie, K.R. (2009) Single Molecule Detection in Nanofluidic Digital Array Enables Accurate Measurement of DNA Copy Number. Analytical and Bioanalytical Chemistry, 394, 457-467.
[25] Baker, M. (2012) Digital PCR Hits Its Stride. Nature Methods, 9, 541-544.
[26] Day, E., Dear, P.H. and McCaughan, F. (2013) Digital PCR Strategies in the Development and Analysis of Molecular Biomarkers for Personalized Medicine. Methods, 59, 101-107.
[27] Racki, N., Dreo, T., Gutierrez-Aguirre, I., Blejec, A. and Ravnikar, M. (2014) Reverse Transcriptase Droplet Digital PCR Shows High Resilience to PCR Inhibitors from Plant, Soil and Water Samples. Plant Methods, 10, 42.
[28] Iwobi, A., Gerdes, L., Busch, U. and Pecoraro, S. (2016) Droplet Digital PCR for Routine Analysis of Genetically Modified Foods (GMO)—A Comparison with Real-Time Quantitative PCR. Food Control, 69, 205-213.
[29] Pohl, G. and Shih, I.M. (2004) Principle and Applications of Digital PCR. Expert Review of Molecular Diagnostics, 4, 41-47.
[30] Burns, M.J., Burrell, A.M. and Foy, C. (2010) The Applicability of Digital PCR for the Assessment of Detection Limits in GMO Analysis. European Food Research and Technology, 231, 353-362.
[31] Devonshire, A.S., Sanders, R., Wilkes, T.M., Taylor, M.S., Foy, C.A. and Huggett, J.F. (2013) Application of Next Generation qPCR and Sequencing Platforms to mRNA Biomarker Analysis. Methods, 59, 89-100.
[32] Morisset, D., Stebih, D., Milavec, M., Gruden, K. and Zel, J. (2013) Quantitative Analysis of Food and Feed Samples with Droplet Digital PCR. PLoS ONE, 8, e62583.
[33] Corbisier, P., Bhat, S., Partis, L., Xie, V. and Emslie, K. (2010) Absolute Quantification of Genetically Modified MON810 Maize (Zea mays L.) by Digital Polymerase Chain Reaction. Analytical and Bioanalytical Chemistry, 396, 2143-2150.
[34] Lievens, A., Jacchia, S., Kagkli, D., Savini, C. and Querci, M. (2016) Measuring Digital PCR Quality: Performance Parameters and Their Optimization. PLoS ONE, 11, e0153317.
[35] Miliaras, N. (2014) Digital PCR Comes of Age. Genetic Engineering & Biotechnology News, 34, 14-16.
[36] Dong, L., Meng, Y., Sui, Z., Wang, J., Wu, L. and Fu, B. (2015) Comparison of Four Digital PCR Platforms for Accurate Quantification of DNA Copy Number of a Certified Plasmid DNA Reference Material. Scientific Reports, 5, Article Number: 13174.
[37] Vaïtilingom, M., Pijnenburg, H., Gendre, F. and Brignon, P. (1999) Real Time of Genetically Modified Maximizer Maize and Roundup Ready Soybean in Some Representative Foods. Journal of Agricultural and Food Chemistry, 47, 5261-5266.
[38] Nair, R.S., Fuchs, R.L. and Schuette, S.A. (2002) Current Methods for Assessing Safety of Genetically Modified Crops as Exemplified by Data on Roundup Ready Soybeans. Toxicologic Pathology, 30, 117-125.
[39] Terry, C.F., Harris, N. and Parkes, H. (2002) Detection of Genetically Modified Crops and Their Derivatives: Critical Steps in Sample Preparation and Extraction. Journal of AOAC International, 85, 768-774.
[40] Rott, M.E., Lawrence, T.S., Wall, E.M. and Green, M.J. (2004) Detection and Quantification of Roundup Ready Soy in Foods by Conventional and Real-Time Polymerase Chain Reaction. Journal of Agricultural and Food Chemistry, 52, 5223-5232.
[41] Andersen, C.B., Holst-Jensen, A., Berdal, K.G., Thorstensen, T. and Tengs, T. (2006) Equal Performance of TaqMan, MGB, Molecular Beacon, and SYBR Green-Based Detection Assays in Detection and Quantification of Roundup Ready Soybean. Journal of Agricultural and Food Chemistry, 54, 9658-9663.
[42] Samson, M.C., Gullì, M. and Marmiroli, N. (2010) Quantitative Detection Method for Roundup Ready Soybean in Food Using Duplex Real-time PCR MGB Chemistry. Journal of the Science of Food and Agriculture, 90, 1437-1444.
[43] Kodama, T., Kasahara, M., Minegishi, Y., Futo, S., Sawada, C., Watai, M., et al. (2011) Qualitative PCR Method for Roundup Ready Soybean: Interlaboratory Study. Journal of AOAC International, 94, 224-231.
[44] Xiao, X., Wu, H., Zhou, X., Xu, S., He, J., Shen, W., et al. (2012) The Combination of Quantitative PCR and Western Blot Detecting CP4-EPSPS Component in Roundup Ready Soy Plant Tissues and Commercial Soy-Related Foodstuffs. Journal Food Science, 77, C603- C608.
[45] European Commission (EC) (2003) Regulation No 1830/2003 of the European Parliament and of the Council of 22 September 2003 Concerning the Traceability and Labelling of Genetically Modified Organisms and the Traceability of Food and Feed Products Produced from GMOs and Amending Directive 2001/18/EC. Official Journal of the European Union, 268, 24-28.
[46] Barbosa, E.G.G., Leite, J.P., Marin, S.R.R., Marinho, J.P., de Fátima Corrêa Carvalho, J., Fuganti-Pagliarini, R., et al. (2013) Overexpression of the ABA-Dependent AREB1 Transcription Factor from Arabidopsis Thaliana Improves Soybean Tolerance to Water Deficit. Plant Molecular Biology Reporter, 31, 719-730.
[47] Demeke, T. and Jenkins, G.R. (2010) Influence of DNA Extraction Methods, PCR Inhibitors and Quantification Methods on Real-Time PCR Assay of Biotechnology-Derived Traits. Analytical and Bioanalytical Chemistry, 396, 1977-1990.
[48] Turkec, A., Kazan, H., Baykut, A. and Lucas, S.J. (2014) Evaluation of DNA Extraction Methods in Order to Monitor GM Materials in Soy Foodstuffs and Feeds Commercialised in Turkey by Multiplex Real-Time PCR. Journal of the Science of Food and Agriculture, 95, 386-392.

comments powered by Disqus

Copyright © 2017 by authors and Scientific Research Publishing Inc.

Creative Commons License

This work and the related PDF file are licensed under a Creative Commons Attribution 4.0 International License.