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Collaborative trial to assess the performance of digital PCR in the field of GMO analysis using an artificial sample material

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Abstract

Digital PCR promises absolute quantification of amplifiable nucleic acids molecules. In consequence, results should be comparable, irrespective of the analysing laboratory and the equipment used. To assess this claim, we used a multitarget DNA molecule (amplicon) encoding for eight transgene soy traits and the lectin soy reference gene. This multitarget amplicon was analysed by seven European laboratories by duplex digital PCR assays. The results showed that absolute measurements of target copy number range revealed high variation. Nevertheless, the measured proportion of the transgene target sequences to the lectin reference gene of the amplicon was in most cases close to 1:1. Digital PCR seems to produce interlaboratory highly reproducible results of relative quantification. Care has to be taken to assess the performance of the applied PCR system. For this purpose, multitarget amplicons may be a valuable amendment to reference material gained from plant material, particularly in cases where reference material is unavailable.

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References

  1. Trapmann S, Corbisier P, Schimmel H, Emons H (2010) Towards future reference systems for GM analysis. Anal Bioanal Chem 396(6):1969–1975

    Article  CAS  Google Scholar 

  2. Sanders R, Huggett JF, Bushell CA, Cowen S, Scott DJ, Foy CA (2011) Evaluation of Digital PCR for Absolute DNA Quantification;Anal. Chem. 83(17):6474–6484

    CAS  Google Scholar 

  3. Pinheira LB, Coleman VA, Hindson CM, Herrmann J, Hindson BJ, Bhat S, Emsli KR (2012) Evaluation of a Droplet Digital Polymerase Chain Reaction Format for DNA copy number quantification Anal. Chem. 84:1003–1011

    Google Scholar 

  4. Hindson CM, Chevillet JR, Briggs HA, Gallichotte EN, Rufa IK, Benjamin J, Hindson R, Vessella L, Tewari M (2013) Absolute quantification by droplet digital PCR versus analog real-time PCR. Nat Methods 10:1003–1005

    Article  CAS  Google Scholar 

  5. Gürtler P, Gerdes L (2014) Digitale Polymerasekettenreaktion, ddPCR; BIOspektrum| 06.14| 20. Jahrgang pp 632–635

  6. Miotke L, Lau BT, Rumma TR, Hanlee P, Ji HP (2014) High sensitivity detection and quantitation of DNA copy number and single nucleotide variants with single color droplet digital PCR. Anal Chem 86(5):2618–2624

    Article  CAS  Google Scholar 

  7. Burns MJ, Burrell AM, Foy CA (2010) The applicability of digital PCR for the assessment of detection limits in GMO analysis. Eur Food Res Technol 231(3):353–362

    Article  CAS  Google Scholar 

  8. Morisset D, Stebih D, Milavec M, Gruden K, Zel J (2013) Quantitative analysis of food and feed samples with droplet digital PCR. PLoS ONE 8(5):e62583

    Article  CAS  Google Scholar 

  9. Milavec M, Dobnik D, Yang L, Zhang D, Gruden K, Zel J (2014) GMO quantification: valuable experience and insights for the future. Anal Bioanal Chem 406(26):6485–6497

    Article  CAS  Google Scholar 

  10. Scholdberg TA, Norden TD, Nelson D, Jenkins RG (2009) Evaluating precision and accuracy when quantifying different endogenous control, reference genes in maize using real-time PCR. J Agric Food Chem 57(7):2903–2911

    Article  CAS  Google Scholar 

  11. Höhne M, Santisi C, Meyer R (2002) Real-time multiplex PCR: an accurate method for the detection and quantification of 35S-CaMV promoter in genetically modified maize-containing food. Eur Food Res Technol 215(1):59–64

    Article  Google Scholar 

  12. Corbisier P, Broothaerts W, Gioria S, Schimmel H, Burns M, Baoutina A, Emslie KR, Furui S, Kurosawa Y, Holden MJ, Kim HH, Lee YM, Kawaharasaki M, Sin D, Wang J (2007) Toward metrological traceability for DNA fragment ratios in GM quantification. 1. Effect of DNA extraction methods on the quantitative determination of Bt176 corn by real-time PCR. J Agric Food Chem 55(9):3249–3257

    Article  CAS  Google Scholar 

  13. Corbisier P, Bhat S, Partis L, Rui V, Xie D, Emslie KR (2010) Absolute quantification of genetically modified MON810 maize (Zea mays L.) by digital polymerase chain reaction. Anal Bioanal Chem 396(6):2143–2150

    Article  CAS  Google Scholar 

  14. Mattarucchi E, Weighardt F, Barbati C, Querci M, Van den Eede G (2005) Development and applications of real-time PCR standards for GMO quantification based on tandem-marker plasmids. Eur Food Res Technol 221(3–4):511–519

    Article  CAS  Google Scholar 

  15. Taverniers I, Windels P, Vaïtilingom M, Milcamps A, Van Bockstaele E, Van den Eede G, De Loose M (2005) Event-specific plasmid standards and real-time PCR methods for transgenic Bt11, Bt176, and GA21 maize and transgenic GT73 canola. J Agric Food Chem 53(8):3041–3052

    Article  CAS  Google Scholar 

  16. Mano J, Hatano S, Futo S, Yoshii J, Nakae H, Naito S, Takabatake R, Kitta K (2014) Development of a reference material of a single DNA molecule for the quality control of PCR testing. Anal Chem 86(17):8621–8627

    Article  CAS  Google Scholar 

  17. Köppel R, Bucher T (2015) Rapid establishment of droplet digital PCR for quantitative GMO analysis. Eur Food Res Technol 241(3):427–439

    Article  Google Scholar 

  18. Trypsteen W, Vynck M, De Neve J, Bonczkowski P, Kiselinova M, Malatinkova E, Vervisch K, Thas O, Vandekerckhove L, De Spiegelaere W (2015) ddpcRquant: threshold determination for single channel droplet digital PCR experiments. Anal Bioanal Chem 407(19):5827–5834

    Article  CAS  Google Scholar 

  19. Application guide of the QX200 manual (2014) p 30

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Acknowledgements

We thank Biosmart GmbH, Switzerland, for providing the artificial multitarget amplicon, used in this study and the Cantonal Laboratory of Zürich for providing the resources for this work. Special thanks go to the participating laboratories for their important work.

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Correspondence to René Köppel.

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Köppel, R., Peterseil, V., Dagand, E. et al. Collaborative trial to assess the performance of digital PCR in the field of GMO analysis using an artificial sample material. Eur Food Res Technol 243, 1091–1096 (2017). https://doi.org/10.1007/s00217-016-2824-8

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  • DOI: https://doi.org/10.1007/s00217-016-2824-8

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