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Cloned plasmid DNA fragments as calibrators for controlling GMOs: different real-time duplex quantitative PCR methods

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Abstract

Analytical real-time PCR technology is a powerful tool for implementation of the GMO labeling regulations enforced in the EU. The quality of analytical measurement data obtained by quantitative real-time PCR depends on the correct use of calibrator and reference materials (RMs). For GMO methods of analysis, the choice of appropriate RMs is currently under debate. So far, genomic DNA solutions from certified reference materials (CRMs) are most often used as calibrators for GMO quantification by means of real-time PCR. However, due to some intrinsic features of these CRMs, errors may be expected in the estimations of DNA sequence quantities. In this paper, two new real-time PCR methods are presented for Roundup Ready soybean, in which two types of plasmid DNA fragments are used as calibrators. Single-target plasmids (STPs) diluted in a background of genomic DNA were used in the first method. Multiple-target plasmids (MTPs) containing both sequences in one molecule were used as calibrators for the second method. Both methods simultaneously detect a promoter 35S sequence as GMO-specific target and a lectin gene sequence as endogenous reference target in a duplex PCR. For the estimation of relative GMO percentages both “delta CT” and “standard curve” approaches are tested. Delta CT methods are based on direct comparison of measured CT values of both the GMO-specific target and the endogenous target. Standard curve methods measure absolute amounts of target copies or haploid genome equivalents. A duplex delta CT method with STP calibrators performed at least as well as a similar method with genomic DNA calibrators from commercial CRMs. Besides this, high quality results were obtained with a standard curve method using MTP calibrators. This paper demonstrates that plasmid DNA molecules containing either one or multiple target sequences form perfect alternative calibrators for GMO quantification and are especially suitable for duplex PCR reactions.

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References

  1. Anklam E, Gadani F, Heinze P, Pijnenburg H, Van den Eede G (2002) Eur Food Res Technol 215:334–339

    Article  CAS  Google Scholar 

  2. Bonfini L, Heinze P, Kay S, Van den Eede G (2002) Review of GMO detection and quantification techniques. IHCP, JRC, EC, EUR 20384 EN, p 67

  3. Griffiths K, Partis L, Croan D, Wang N, Emslie K (2002) Review of technologies for detecting genetically modified materials in commodities and food. Department of Agriculture, Fisheries and Forestry, Australian Government Analytical Laboratories (AGAL), p 118

  4. Holst-Jensen A, Ronning SB, Lovseth A, Berdal KG (2003) Anal Bioanal Chem 375:985–993

    CAS  PubMed  Google Scholar 

  5. European Commission Regulation 1829/2003 Off J Eur Communities L 268/1; 18.10.2003

  6. European Commission Regulation 258/97 Off J Eur Communities L 43; 14.2.1997

  7. http://www.europa.eu.int, MEMO/02/160-REV, Brussels. Cited 4 Mar 2003

  8. Walker R, Lumley I (1999) Trends Anal Chem 18:594–616

    Article  CAS  Google Scholar 

  9. Pietsch K, Waiblinger HU (1999) In: Meuer S, Wittwer C, Nakagawara K (eds) Rapid cycle real-time PCR—methods and applications, preprint, p 7

  10. Wurz A, Bluth A, Zeltz P, Pfeifer C, Willmund R (1999) Food Control 10:385–389

    Article  Google Scholar 

  11. Alary R, Serin A, Maury D, Jouira HB, Sirven J-P, Gautier M-F, Joudrier P (2002) Food Control 13:235-244

    Google Scholar 

  12. Zeitler R, Pietsch K, Waiblinger HU (2002) Eur Food Res Technol 214:346–351

    Article  CAS  Google Scholar 

  13. Dahinden I, Zimmermann A, Liniger M, Pauli U (2002) In: Reischl U, Wittwer C, Cockerill F (eds) Rapid cycle real-time PCR—methods and applications, microbiology and food analysis. Springer, Berlin Heidelberg New York, pp 251–258

  14. Brodmann PD, Ilg EC, Berthoud H, Herrmann A (2002) J AOAC Int 85:646–653

    CAS  PubMed  Google Scholar 

  15. Hübner P, Waiblinger H-U, Pietsch K (2001) J AOAC Int 84:1855–1864

    CAS  PubMed  Google Scholar 

  16. Terzi V, Farrari B, Finocchiaro F, De Fonze N, Stanca AM, Lamacchia C, Napier J, Shewry AR, Faccioli P (2003) J Cereal Sci 37:157–163

    Article  CAS  Google Scholar 

  17. Block A, Schwarz G (2003) Eur Food Res Technol 216:421–427

    CAS  Google Scholar 

  18. Vaïtilingom M, Pijnenburg H, Gendre F, Brignon P (1999) J Agric Food Chem 47:5261–5266

    PubMed  Google Scholar 

  19. Berdal KG, Holst-Jensen A (2001) Eur Food Res Technol 213:432–438

    CAS  Google Scholar 

  20. Hernandez M, Pla M, Esteve T, Prat S, Puigdomenech P, Ferrando A (2003) Transgenic Res 12:179–189

    Article  CAS  PubMed  Google Scholar 

  21. Ronning SB, Vaïtilingom M, Berdal KG, Holst-Jensen A (2003) Eur Food Res Technol 216:347–354

    CAS  Google Scholar 

  22. Terry CF, Shanahan DJ, Ballam LD, Harris N, McDowell DG, Parkes HC (2002) J AOAC Int 85:938–944

    CAS  PubMed  Google Scholar 

  23. Hird H, Powell J, Johnson M-L, Oehlschlager S (2003) J AOAC Int 86:66–71

    CAS  PubMed  Google Scholar 

  24. Terry CF, Harris N (2001) Eur Food Res Technol 213:425–431

    CAS  Google Scholar 

  25. Pardigol A, Guillet S, Pöpping B (2003) Eur Food Res Technol 216:412–420

    CAS  Google Scholar 

  26. Holck A, Vaïtilingom M, Didierjean L, Rudi K (2002) Eur Food Res Technol 214:449–453

    CAS  Google Scholar 

  27. Taverniers I, Windels P, Van Bockstaele E, De Loose M (2001) Eur Food Res Technol 213:417–424

    CAS  Google Scholar 

  28. 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 85:1077–1089

    CAS  PubMed  Google Scholar 

  29. Trapmann S, Catalani P, Conneely P, Corbisier P, Gancberg D, Hannes E, Le Guern L, Kramer GN, Prokisch J, Robouch P, Schimmel H, Zeleny R, Pauwels J, Van den Eede G, Weighardt F, Mazzara M, Anklam E (2002) EUR 19573, European Commission

  30. Shindo Y, Kuribara H, Matsuoka T, Futo S, Sawada C, Shono J, Akiyama H, Goda Y, Toyoda M, Hino A (2002) J AOAC Int 85:1119–1126

    CAS  PubMed  Google Scholar 

  31. Windels P, Theuns I, Dendauw J, Depicker A, Van Bockstaele E, De Loose M (1999) Med Fac Landbouww Univ Gent 64:459–462

    CAS  Google Scholar 

  32. Windels P, Taverniers I, Depicker A, Van Bockstaele E, De Loose M (2001) Eur Food Res Technol 213:107–112

    Article  CAS  Google Scholar 

  33. Puumalainen J, Paoletti C, Bertheau Y, Bonner P, Broll H, Damant A, De Santis B, Holst-Jensen A, Laurensse E, Monsted Jorgensen M, Onori R, Stephensen Lübeck P, Taverniers I, Trapmann S, Van den Eede G (2003) Definition of minimum performance requirements for analytical methods of GMO testing, ENGL WG Validation, Version 1.7.2003

  34. Burns M, Shanahan D, Valdivia H, Harris N (2003) Eur Food Res Technol 216:428–433

    CAS  Google Scholar 

  35. Anklam E, Neumann, DA (2002) J AOAC Int 85:754–756

    CAS  PubMed  Google Scholar 

  36. Van den Eede G, Kay S, Anklam E (2002) J AOAC Int 85:757–761

    PubMed  Google Scholar 

  37. Theuns I, Windels P, De Buck S, Depicker A, Van Bockstaele E, De Loose M (2002) Euphytica 123:75–84

    Article  CAS  Google Scholar 

  38. Taverniers et al. (in preparation)

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Acknowledgements

We wish to thank Cindy Merckaert and Friedle Vanhee for excellent technical assistance, and Didier Allaer from Diagenode for providing the GM Soybean (RRS) Detection Plasmid Set. This work was financially supported by DWTC and O&O from the Belgian Government and by the EU.

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Authors and Affiliations

  1. Department for Plant Genetics and Breeding (DVP-CLO), Ministry of the Flemish Community, Caritasstraat 21, 9090, Melle, Belgium

    Isabel Taverniers, Erik Van Bockstaele & Marc De Loose

  2. Department of Plant Production, Ghent University, Coupure Links 653, 9000, Gent, Belgium

    Erik Van Bockstaele

Authors
  1. Isabel Taverniers
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  2. Erik Van Bockstaele
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  3. Marc De Loose
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Correspondence to Isabel Taverniers.

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Taverniers, I., Van Bockstaele, E. & De Loose, M. Cloned plasmid DNA fragments as calibrators for controlling GMOs: different real-time duplex quantitative PCR methods. Anal Bioanal Chem 378, 1198–1207 (2004). https://doi.org/10.1007/s00216-003-2372-5

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  • DOI: https://doi.org/10.1007/s00216-003-2372-5

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