Analytical and Bioanalytical Chemistry

, Volume 396, Issue 6, pp 2073–2089

Molecular toolbox for the identification of unknown genetically modified organisms

  • Tom Ruttink
  • Rolinde Demeyer
  • Elke Van Gulck
  • Bart Van Droogenbroeck
  • Maddalena Querci
  • Isabel Taverniers
  • Marc De Loose
Original Paper

Abstract

Competent laboratories monitor genetically modified organisms (GMOs) and products derived thereof in the food and feed chain in the framework of labeling and traceability legislation. In addition, screening is performed to detect the unauthorized presence of GMOs including asynchronously authorized GMOs or GMOs that are not officially registered for commercialization (unknown GMOs). Currently, unauthorized or unknown events are detected by screening blind samples for commonly used transgenic elements, such as p35S or t-nos. If (1) positive detection of such screening elements shows the presence of transgenic material and (2) all known GMOs are tested by event-specific methods but are not detected, then the presence of an unknown GMO is inferred. However, such evidence is indirect because it is based on negative observations and inconclusive because the procedure does not identify the causative event per se. In addition, detection of unknown events is hampered in products that also contain known authorized events. Here, we outline alternative approaches for analytical detection and GMO identification and develop new methods to complement the existing routine screening procedure. We developed a fluorescent anchor-polymerase chain reaction (PCR) method for the identification of the sequences flanking the p35S and t-nos screening elements. Thus, anchor-PCR fingerprinting allows the detection of unique discriminative signals per event. In addition, we established a collection of in silico calculated fingerprints of known events to support interpretation of experimentally generated anchor-PCR GM fingerprints of blind samples. Here, we first describe the molecular characterization of a novel GMO, which expresses recombinant human intrinsic factor in Arabidopsis thaliana. Next, we purposefully treated the novel GMO as a blind sample to simulate how the new methods lead to the molecular identification of a novel unknown event without prior knowledge of its transgene sequence. The results demonstrate that the new methods complement routine screening procedures by providing direct conclusive evidence and may also be useful to resolve masking of unknown events by known events.

Figure

Molecular toolbox for the identification of genetically modified organisms

Keywords

GMO screening analysis GMO identification Anchor-PCR Fingerprinting p35S t-nos 

Supplementary material

216_2009_3287_MOESM1_ESM.pdf (1.9 mb)
ESM 1(PDF 1957 kb)

References

  1. 1.
    James C (2009) ISAAA brief 39Google Scholar
  2. 2.
    Stein AJ, Rodríguez-Cerezo E (2009) European Communities, Luxembourg, JRC Technical Report EUR 23486 ENGoogle Scholar
  3. 3.
    Stein AJ, Rodríguez-Cerezo E (2009) European Communities, Luxembourg, JRC Technical Report EUR 23810 ENGoogle Scholar
  4. 4.
    Community Reference Laboratory for GM Food and Feed (2009) . Status of dossiers. http://gmo-crl.jrc.ec.europa.eu/statusofdoss.htm. Accessed 30 Sept 2009
  5. 5.
    Dorey E (2000) Nat Biotechnol 18:1136–1137CrossRefGoogle Scholar
  6. 6.
    European Commission (2005) Off J Eur Union 101:14–16Google Scholar
  7. 7.
    European Commission (2006) Off J Eur Union 306:17–20Google Scholar
  8. 8.
    European Commission (2008) Off J Eur Union 96:29–34Google Scholar
  9. 9.
    Vermij P (2006) Nat Biotechnol 24:1301–1302CrossRefGoogle Scholar
  10. 10.
    Holst-Jensen A (2008) Nat Biotechnol 26:858–859CrossRefGoogle Scholar
  11. 11.
    Ruttink T, Morisset D, Van Droogenbroeck B, Lavrac N, Van Den Eede G, Zel J, De Loose M (2009) Anal Bioanal Chem. doi:10.1007/s00216-009-3218-6
  12. 12.
  13. 13.
    Kuribara H, Shindo Y, Matsuoka T, Takubo K, Futo S, Aoki N, Hirao T, Akiyama H, Goda Y, Toyoda M, Hino A (2002) J AOAC Int 85:1077–1189Google Scholar
  14. 14.
    Hernandez M, Rodriguez-Lazaro D, Ferrando A (2005) Curr Anal Chem 1:203–221CrossRefGoogle Scholar
  15. 15.
    Xu J, Miao H, Wu H, Huang W, Tang R, Qiu M, Wen J, Zhu S, Li Y (2006) Biosens Bioelectron 22:71–77CrossRefGoogle Scholar
  16. 16.
    Holst-Jensen A, Rønning SB, Løvseth A, Berdal KG (2003) Anal Bioanal Chem 375:985–993Google Scholar
  17. 17.
    Leimanis S, Hernández M, Fernández S, Boyer F, Burns M, Bruderer S, Glouden T, Harris N, Kaeppeli O, Philipp P, Pla M, Puigdomènech P, Vaitilingom M, Bertheau Y, Remacle J (2006) Plant Mol Biol 61:123–139CrossRefGoogle Scholar
  18. 18.
    Prins TW, van Dijk JP, Beenen HG, Van Hoef AA, Voorhuijzen MM, Schoen CD, Aarts HJ, Kok EJ (2008) BMC Genomics 9:584CrossRefGoogle Scholar
  19. 19.
    Schmidt AM, Sahota R, Pope DS, Lawrence TS, Belton MP, Rott ME (2008) J Agric Food Chem 56:6791–6800CrossRefGoogle Scholar
  20. 20.
    Chaouachi M, Chupeau G, Berard A, McKhann H, Romaniuk M, Giancola S, Laval V, Bertheau Y, Brunel D (2008) J Agric Food Chem 56:11596–11606CrossRefGoogle Scholar
  21. 21.
    Randhawa GJ, Chhabra R, Singh M (2009) J Agric Food Chem 57:5167–5172CrossRefGoogle Scholar
  22. 22.
    Mano J, Shigemitsu N, Futo S, Akiyama H, Teshima R, Hino A, Furui S, Kitta K (2009) J Agric Food Chem 57:26–37CrossRefGoogle Scholar
  23. 23.
    Elenis DS, Kalogianni DP, Glynou K, Ioannou PC, Christopoulos TK (2008) Anal Bioanal Chem 392:347–354CrossRefGoogle Scholar
  24. 24.
    Hamels S, Glouden T, Gillard K, Mazzara M, Debode F, Foti N, Sneyers M, Nuez TE, Pla M, Berben G, Moens W, Bertheau Y, Audeon C, Van den Eede G, Remacle J (2009) Eur Food Res Tech 228:531–543CrossRefGoogle Scholar
  25. 25.
    Miraglia M, Berdal KG, Brera C, Corbisier P, Holst-Jensen A, Kok EJ, Marvin HJ, Schimmel H, Rentsch J, van Rie JP, Zagon J (2004) Food Chem Toxicol 42:1157–1180CrossRefGoogle Scholar
  26. 26.
    GMO Compass (2009) http://www.gmo-compass.org. Accessed 30 Sept 2009
  27. 27.
    Cankar K, Chauvensy-Ancel V, Fortabat MN, Gruden K, Kobilinsky A, Žel J, Bertheau Y (2008) Anal Biochem 376:189–199CrossRefGoogle Scholar
  28. 28.
    Raymond P, Gendron L, Paul S, Moreau ME, Coté MJ, Laberge S (2008) In: Book of abstracts. First Global Conference on GMO Analysis, Como, Italy, 24–27 June 2008Google Scholar
  29. 29.
    Bhat NS, Dibley K, Xie VRD, Partis L, Raymond P, Emslie KR (2008) In: Book of abstracts. First Global Conference on GMO Analysis, Como, Italy, 24–27 June 2008Google Scholar
  30. 30.
    Nesvold H, Kristoffersen AB, Holst-Jensen A, Berdal KG (2005) Bioinformatics 21:1917–1926CrossRefGoogle Scholar
  31. 31.
    Querci M, Foti N, Bogni A, Kluga L, Broll H, Van den Eede G (2009) Food Anal. Methods. doi:10.1007/s12161-009-9093-0 Google Scholar
  32. 32.
    Theuns I, Windels P, De Buck S, Depicker A, Van Bockstaele E, De Loose M (2002) Euphytica 123:75–84CrossRefGoogle Scholar
  33. 33.
    Windels P, De Buck S, Van Bockstaele E, De Loose M, Depicker A (2003) Plant Physiol 133:2061–2068CrossRefGoogle Scholar
  34. 34.
    Alonso JM et al (2003) Science 301:653–657CrossRefGoogle Scholar
  35. 35.
    Dong W, Yang L, Shen K, Kim B, Kleter GA, Marvin HJP, Guo R, Liang W, Zhang D (2008) BMC Bioinformatics 9:260CrossRefGoogle Scholar
  36. 36.
    Fedosov SN, Laursen NB, Nexø E, Moestrup SK, Petersen TE, Jensen EØ, Berglund L (2003) Eur J Biochem 270:3362–3367CrossRefGoogle Scholar
  37. 37.
    Hvas AM, Buhl H, Laursen NB, Hesse B, Berglund L, Nexo E (2006) Haematologica 91:805–808Google Scholar
  38. 38.
    GM Database (2009) Agbios. http://www.agbios.com/dbase.php. Accessed 30 Sept 2009
  39. 39.
    Debode F, Janssen E, Berben G (2007) Eur Food Res Tech 226:273–280CrossRefGoogle Scholar
  40. 40.
    GMOtrack (2009) http://kt.ijs.si/software/GMOtrack/. Accessed 30 Sept 2009

Copyright information

© Springer-Verlag 2009

Authors and Affiliations

  • Tom Ruttink
    • 1
  • Rolinde Demeyer
    • 1
  • Elke Van Gulck
    • 2
  • Bart Van Droogenbroeck
    • 1
  • Maddalena Querci
    • 3
  • Isabel Taverniers
    • 1
  • Marc De Loose
    • 1
    • 4
  1. 1.Institute for Agricultural and Fisheries Research (ILVO), Technology and Food Science Unit—Product Quality and InnovationMerelbekeBelgium
  2. 2.KaHo Sint-Lieven, Departement GentGhentBelgium
  3. 3.Molecular Biology and Genomics Unit, European Commission - Joint Research Center (JRC)Institute for Health and Consumer ProtectionIspraItaly
  4. 4.Department of Molecular GeneticsGhent UniversityGhentBelgium

Personalised recommendations