European Food Research and Technology

, Volume 236, Issue 4, pp 599–606 | Cite as

Development of a CTAB buffer-based automated gDNA extraction method for the surveillance of GMO in seed

  • Patrick Guertler
  • Andrea Harwardt
  • Adelina Eichelinger
  • Paul Muschler
  • Ottmar Goerlich
  • Ulrich Busch
Original Paper


Seed imported into the EU from countries growing genetically modified (gm) plants may contain traces of these gm crops. As a result of the zero tolerance policy of the EU, these products must be removed from the market. Along with the amount of biotech crops produced worldwide, the work load for seed surveillance authorities increases. Since the commonly used CTAB buffer-based extraction methods are manual and laborious, a large part of the work load is caused by DNA extraction. In order to reduce labour input and accelerate the DNA analysis workflow, we developed an automated CTAB buffer-based DNA isolation method for seed. Several isolation and chemistry parameters were altered to combine a thorough cell lysis, removal of inhibitors and a highly efficient binding of gDNA to paramagnetic beads. This optimised procedure was compared with manual CTAB buffer-based and Wizard-based DNA extraction methods for maize, soya bean and rapeseed. Automated DNA extraction was faster, less laborious and resulted, on average, in higher DNA yield and purity. The applicability of our method was successfully proven with in-house routine samples.


Automated nucleic acid extraction Maxwell 16 CTAB Genetically modified plants GMO Seed Quantitative real-time PCR 



The authors would like to thank Ingo Engels for his preliminary work. Promega GmbH is particularly acknowledged for providing Maxwell® 16 AS1290 kits.


  1. 1.
    Busch U, Pecoraro S, Posthoff K, Estendorfer-Rinner S (2004) Erster Nachweis einer gentechnisch veränderten Papaya in Europa - Beanstandung eines in der EU nicht zugelassenen gentechnisch veränderten Organismus. Deut Lebensm-Rundsch 100(10):377–380Google Scholar
  2. 2.
    Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, Mueller R, Nolan T, Pfaffl MW, Shipley GL, Vandesompele J, Wittwer CT (2009) The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem 55(4):611–622CrossRefGoogle Scholar
  3. 3.
    Callejas S, Alvarez R, Dopazo A (2011) Automatic Genomics: a user-friendly program for the automatic designing and plate loading of medium-throughput qPCR experiment. Biotechniques 50:46–50CrossRefGoogle Scholar
  4. 4.
    EC (2001) Directive 2001/18/EC of the European Parliament and of the Council on the deliberate release into the environment of genetically modified organisms and repealing Council Directive 90/220/EEC. Off J Eur Un L 106/1Google Scholar
  5. 5.
    EC (2003a) Regulation (EC) No 1829/2003 of the European Parliament and of the Council of 22 September 2003 on genetically modified food and feed. Off J Eur Un L 268/1Google Scholar
  6. 6.
    EC (2003b) 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. Off J Eur Un L 268/24Google Scholar
  7. 7.
    EURL (2012) European Union Reference Laboratory for GM food & feed. 2012
  8. 8.
    Foley C, O’Farrelly C, Meade KG (2011) Technical note: comparative analyses of the quality and yield of genomic DNA from invasive and non-invasive, automated and manual extraction methods. J Dairy Sci 94(6):3159–3165CrossRefGoogle Scholar
  9. 9.
    Frederick R, Bergeman L, Blommel P, Bailey L, McCoy J, Song J, Meske L, Bingman C, Riters M, Dillon N, Kunert J, Yoon J, Lim A, Cassidy M, Bunge J, Aceti D, Primm J, Markley J, Phillips G, Fox B (2007) Small-scale, semi-automated purification of eukaryotic proteins for structure determination. J Struct Funct Genomics 8(4):153–166CrossRefGoogle Scholar
  10. 10.
    Guertler P, Huber I, Pecoraro S, Busch U (2012) Development of an event-specific detection method for genetically modified rice Kefeng 6 by quantitative real-time PCR. J Verbrauch Lebensm 7:63–70CrossRefGoogle Scholar
  11. 11.
    Guertler P, Paul V, Steinke K, Wiedemann S, Preißinger W, Albrecht C, Spiekers H, Schwarz FJ, Meyer HHD (2010) Long-term feeding of genetically modified corn (MON810)—Fate of cry1Ab DNA and recombinant protein during the metabolism of the dairy cow. Livest Sci 131(2–3):250–259CrossRefGoogle Scholar
  12. 12.
    ISO 2157 0:2005/DAM 1:2012 (2005)Google Scholar
  13. 13.
    James C (2011) Global Status of Commercialized Biotech/GM Crops: 2011. ISAAA Brief No. 43:ISAAA Ithaca, NYGoogle Scholar
  14. 14.
    Khokhar SK, Mitui M, Leos Nora K, Rogers Beverly B, Park Jason Y (2012) Evaluation of Maxwell® 16 for automated DNA extraction from whole blood and formalin-fixed paraffin embedded (FFPE) tissue. Clinical Chemistry and Laboratory Medicine, vol 50Google Scholar
  15. 15.
    Kuribara H, Shindo Y, Matsuoka T, Takubo K, Futo S, Aoki N, Hirao T, Akiyama H, Goda Y, Toyoda M, Hino A (2002) Novel reference molecules for quantitation of genetically modified maize and soybean. J AOAC Int 85(5):1077–1089Google Scholar
  16. 16.
    LAG (2012a) German Working Group on Genetic Engineering of the Federal States and the Federal Government (Methodensammlung der Bund/Länder-Arbeitsgemeinschaft Gentechnik), Real-time PCR-Verfahren zum Event-spezifischen Nachweis der Rapslinien Falcon GS40/90 und Liberator pHoe6/Ac.
  17. 17.
    LAG (2012b) German Working Group on Genetic Engineering of the Federal States and the Federal Government (Methodensammlung der Bund/Länder-Arbeitsgemeinschaft Gentechnik), Real-Time PCR zur quantitativen Bestimmung gentechnisch veränderter Rapslinien mit dem 35S/pat-Genkonstrukt.
  18. 18.
    Martín-Núñez GM, Gómez-Zumaquero JM, Soriguer F, Morcillo S (2012) High resolution melting curve analysis of DNA samples isolated by different DNA extraction methods. Clin Chim Acta 413(1–2):331–333CrossRefGoogle Scholar
  19. 19.
    Reiting R, Grohmann L, Mäde D (2010) A testing cascade for the detection of genetically modified rice by real-time PCR in food and its application for detection of an unauthorized rice line similar to KeFeng6. J Verbrauch Lebensm 5(2):185–188CrossRefGoogle Scholar
  20. 20.
    Waiblinger H-U, Grohmann L, Mankertz J, Engelbert D, Pietsch K (2010) A practical approach to screen for authorised and unauthorised genetically modified plants. Anal Bioanal Chem 396(6):2065–2072CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  • Patrick Guertler
    • 1
  • Andrea Harwardt
    • 2
  • Adelina Eichelinger
    • 1
  • Paul Muschler
    • 3
  • Ottmar Goerlich
    • 1
  • Ulrich Busch
    • 1
  1. 1.Bavarian Health and Food Safety AuthorityOberschleißheimGermany
  2. 2.Synlab LabordienstleistungenMunichGermany
  3. 3.Promega GmbHMannheimGermany

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