European Food Research and Technology

, Volume 237, Issue 2, pp 125–135 | Cite as

Evaluation of Adh1 alleles and transgenic soybean seeds using Scorpion PCR and HRM analysis

  • Zita Erika Madi
  • Christian Brandes
  • Georg Neumann
  • David Quist
  • Werner Ruppitsch
  • Rupert Hochegger
Original Paper

Abstract

The identification of both approved and non-approved genetically modified organisms (GMOs) is an integral part of GMO biosafety legislation in many countries. One aspect that may affect PCR-based detection of a GMO lies within the analysis of its genetic stability, as sequence alterations or DNA instabilities may impede quantification by PCR. Genetic stability can be analyzed using various methods, yet many of these methods have distinct disadvantages, including low sensitivity. In this study, high resolution melting (HRM) analysis and real-time PCR with Scorpion primers were used as a method to analyze the 3′ end of RR soybeans (RR 40-3-2) in a large number of samples (n = 1,034). No evidence for the occurrence of mutation events was found, implying that the nucleotide sequence of this region is unlikely to be unstable and is well suited as a target for the quantification of RR soybeans. Additionally, and as a preparative work for an optimization of the method, a 174 bp region of the first intron of the Adh1 gene was analyzed in several varieties of maize with different GMO events using the same approach. The results show that 2 alleles are present. In further experiments, the different alleles were cloned into plasmids to generate homozygous plasmids from heterozygous templates in order to generate for a more precise analysis. The overall methodological aim of these studies was to compare HRM analysis with Scorpion primer PCR. Both methods were capable of differentiating between the 2 homozygous and heterozygous alleles. For a better discrimination, however, we conclude that it is most reliable to consider the results of both methods. This dual approach is assumed to be an effective tool as an accurate, high-throughput means of the screening of GMOs for potential genetic instabilities that may interfere with the detection and identification of specific GM events.

Keywords

GMO Scorpion primer Adh1 HRM 

References

  1. 1.
    Directive 2001/18/EC of the European Parliament and of the Council of 12 March 2001 on the deliberate release into the environment of genetically modified organisms and repealing Council Directive 90/220/EEC–Commission Declaration. OJ L 106, 17.4.2001, pp 1–39Google Scholar
  2. 2.
    REGULATION (EC) No 1829/2003 of the European Parliament and of the Council of 22 September 2003 on genetically modified food and feed. OJ L268, 18.10. 2003, pp 1–23Google Scholar
  3. 3.
    REGULATION (EC) 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 genetically modified organisms and amending Directive 2001/18/EC. OJ L 268, 18.10.2003, pp 24–28Google Scholar
  4. 4.
    Marmiroli N, Maestri E, Gulli M, Malcevscgi A, Peano C, Bordoni R, De Bellis G (2008) Methods for detection of GMOs in food and feed. Anal Bioanal Chem 392(3):369–384CrossRefGoogle Scholar
  5. 5.
    Kluga L, Follini S, Van den Bulcke M, Van den Eede G, Querci M (2012) Applicability of the “real-time PCR-based ready-to-use multi-target analytical system for GMO detection” in processed maize matrices. Eur Food Res Technol 234(1):109–118CrossRefGoogle Scholar
  6. 6.
    Hernández M, Rodríguez-Lázaro D, Ferrando A (2005) Current methodology for detection, identification and quantification of genetically modified organisms. Curr Anal Chem 1:203–221CrossRefGoogle Scholar
  7. 7.
    Forsbach A, Schubert D, Lechtenberg B, Gils M, Schmidt R (2003) A comprehensive characterization of single-copy T-DNA insertions in the Arabidopsis thaliana genome. Plant Mol Biol 52:161–176CrossRefGoogle Scholar
  8. 8.
    Latham JR, Wilson AK, Steinbrecher RA (2006) The mutational consequences of plant transformation. J Biomed Biotechnol 2006:1–7Google Scholar
  9. 9.
    Wilson A, Latham J, Steinbrecher RA (2006) Transformation induced mutations in transgenic plants: analysis and biosafety implications. Biotechnol Genet Eng Rev 23:209–234CrossRefGoogle Scholar
  10. 10.
    Smith N, Kilpatrick JB, Whitelam GC (2001) Superfluous transgene integration in plants. Crit Rev Plant Sci 20:215–249Google Scholar
  11. 11.
    Belgian Biosafety Server (2006) Molecular characterisation of the genetic maps of commercial modified plants. http://www.biosafety.be/gmcropff/TP/MGC.html
  12. 12.
    Nacry P, Camilleri Ch, Courtial B, Caboche M, Bouchez D (1998) Major chromosomal rearrangements induced by T-DNA transformation in Arabidopsis. Genetics 149(2):641–650Google Scholar
  13. 13.
    Windels P, De Buck S, Van Bockstaele E, De Loose M, Depicker A (2003) T-DNA integration in Arabidopsis chromosomes. Presence and origin of filler DNA sequences. Plant Physiol 133(4):2061–2068CrossRefGoogle Scholar
  14. 14.
    McCabe MS, Mohapatra UB et al (1999) Integration, expression and inheritance of two linked T-DNA marker genes in transgenic lettuce. Mol Breed 5(4):329–344CrossRefGoogle Scholar
  15. 15.
    Ogasawara T, Chikagawa Y, Arakawa F, Nozaki A, Itoh Y, Sasaki K, Umetsu H, Watanabe T, Akiyama H, Maitani T, Toyoda M, Kamada H, Goda Y, Ozeki Y (2005) Frequency of mutations of the transgene, which might result in the loss of the glyphosate-tolerant phenotype, was lowered in Roundup Ready soybeans. J Health Sci 51:197–201CrossRefGoogle Scholar
  16. 16.
    Padgette SR, Kolacz KH, Delannay X, Re DB, Lavallee BJ, Tinius CN, Rhodes WK, Otero YI, Barry GF, Eichholtz DA, Peschke VM, Nida DL, Taylor NB, Kishore GM (1995) Development, identification, and characterization of a glyphosate-tolerant soybean line. Crop Sci 35:1451–1461CrossRefGoogle Scholar
  17. 17.
    Fearing PL, Brown D, Vlachos D, Meghji M, Privalle L (1997) Quantitative analysis of CryIA(b) expression in Bt maize plants, tissues, and silage and stability of expression over successive generations. Mol Breed 3:169–176CrossRefGoogle Scholar
  18. 18.
    Nguyen HT, Jehle JA (2007) Quantitative analysis of the seasonal and tissue-specific expression of Cry1Ab in transgenic maize Mon810. J Plant Dis Prot 114(2):82–87Google Scholar
  19. 19.
    Rosati A, Bogani P, Santarlasci A, Buiatti M (2008) Characterisation of 3_ transgene insertion site and derived mRNAs in MON810 YieldGard maize. Plant Mol Biol 67:271–281CrossRefGoogle Scholar
  20. 20.
    La Paz JL, Pla M, Papazova N, Puigdomènech P, Vicient CM (2010) Stability of the MON 810 transgene in maize. Plant Mol Biol 74(6):563–571CrossRefGoogle Scholar
  21. 21.
    Choffnes DS, Philip R, Vodkin LO (2001) A transgenic locus in soybean exhibits a high level of recombination. In Vitro Cell Dev Biol Plant 37:756–762CrossRefGoogle Scholar
  22. 22.
    Morisset D, Demsar T, Gruden K, Vojvoda J, Stebih D, Zel J (2009) Detection of genetically modified organisms–closing the gaps. Nat Biotechnol 27(8):700–701CrossRefGoogle Scholar
  23. 23.
    Zaulet M, Rusu L, Kevorkian S, Luca C, Mihacea S, Badea EM, Costache M (2009) Detection and quantification of GMO and sequencing of the DNA amplified products. Rom Biotechnol Lett 14(5):4733–4746Google Scholar
  24. 24.
    Mason G, Provero P, Vaira AM, Accotto GP (2002) Estimating the number of integrations in transformed plants by quantitative realtime PCR. BMC Biotechnol 2:20CrossRefGoogle Scholar
  25. 25.
    Broothaerts W, Corbisier P, Schimmel H, Trapmann S, Vincent S, Emons H (2008) A single nucleotide polymorphism (SNP839) in the adh1 reference gene affects the quantitation of genetically modified maize (Zea mays L.). J Agric Food Chem 56(19):8825–8831CrossRefGoogle Scholar
  26. 26.
    Yeung AT, Hattangadi D, Blakesley L, Nicolas E (2005) Enzymatic mutation detection technologies. Biotechniques 38:749–758CrossRefGoogle Scholar
  27. 27.
    Bush SM, Krysan PJ (2010) iTILLING: a personalized approach to the identification of induced mutations in Arabidopsis. Plant Physiol 154:25–35CrossRefGoogle Scholar
  28. 28.
    Papazova N, Ghedira R, Van Glabeke S, Bartegi A, Windels P, Taverniers I, Roldan-Ruiz I, Van Bockstaele E, Milcamps A, Van Den Eede G, Depicker A, De Loose M (2008) Stability of the T-DNA flanking regions in transgenic Arabidopsis thaliana plants under influence of abiotic stress and cultivation practices. Plant Cell Rep 27:749–757CrossRefGoogle Scholar
  29. 29.
    Dong C, Vincent K, Sharp P (2009) Simultaneous mutation detection of three homologous genes in wheat by high resolution melting analysis and mutation surveyor. BMC Plant Biol 9:143–155CrossRefGoogle Scholar
  30. 30.
    Solinas A, Brown LJ, McKeen C, Mellor JM, Nicol J, Thelwell N, Brown T (2001) Duplex Scorpion primers in SNP analysis and FRET applications. Nucleic Acids Res 29:1–9CrossRefGoogle Scholar
  31. 31.
    Thelwell N, Millington S, Solinas A, Booth J, Brown T (2000) Mode of action and application of Scorpion primers to mutation detection. Nucleic Acids Res 28(19):3752–3761CrossRefGoogle Scholar
  32. 32.
    Neumann G, Brandes Ch, Joachimsthaler A, Hochegger R (2011) Assessment of the genetic stability of GMOs with a detailed examination of MON810 using Scorpion probes. Eur Food Res Technol 233(1):19–30CrossRefGoogle Scholar
  33. 33.
  34. 34.
    Aguilera M, Querci M, Balla B, Prospero A, Ermolli M, Van den Eede G (2008) A qualitative approach for the assessment of the genetic stability of the MON 810 trait in commercial seed maize varieties. Food Anal Methods 1:252–258CrossRefGoogle Scholar
  35. 35.
    Aguilera M, Querci M, Pastor-Benito S, Bellocchi G, Milcamps A, Van den Eede G (2009) Assessing copy number of MON 810 integrations in commercial seed maize varieties by 5_ eventspecific real-time PCR validated method coupled to 2_CT analysis. Food Anal Methods 2:73–79CrossRefGoogle Scholar
  36. 36.
    EFSA (2006) Guidance document for the risk assessment of genetically modified plants and derived food and feed. EFSA J 99:1–100Google Scholar
  37. 37.
    GMO Detection method Database (GMDD) http://gmdd.shgmo.org/primer/view/eventspecific/2356

Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  • Zita Erika Madi
    • 1
  • Christian Brandes
    • 1
  • Georg Neumann
    • 4
  • David Quist
    • 2
  • Werner Ruppitsch
    • 3
  • Rupert Hochegger
    • 1
  1. 1.Austrian Agency for Health and Food Safety, Department for Molecular Biology and MicrobiologyInstitute for Food Safety ViennaViennaAustria
  2. 2.GenØk—Centre for BiosafetyTromsoeNorway
  3. 3.Austrian Agency for Health and Food Safety, Department for Medical Microbiology and HygieneCore Unit Molecular BiologyViennaAustria
  4. 4.Baxter Innovations GmbHViennaAustria

Personalised recommendations