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
Trapmann S, Corbisier P, Schimmel H, Emons H (2010) Towards future reference systems for GM analysis. Anal Bioanal Chem 396(6):1969–1975
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
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
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
Gürtler P, Gerdes L (2014) Digitale Polymerasekettenreaktion, ddPCR; BIOspektrum| 06.14| 20. Jahrgang pp 632–635
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
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
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
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
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
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
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
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
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
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
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
Köppel R, Bucher T (2015) Rapid establishment of droplet digital PCR for quantitative GMO analysis. Eur Food Res Technol 241(3):427–439
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
Application guide of the QX200 manual (2014) p 30
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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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