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Analytical and Bioanalytical Chemistry

, Volume 396, Issue 6, pp 2143–2150 | Cite as

Absolute quantification of genetically modified MON810 maize (Zea mays L.) by digital polymerase chain reaction

  • Philippe CorbisierEmail author
  • Somanath Bhat
  • Lina Partis
  • Vicki Rui Dan Xie
  • Kerry R. Emslie
Original Paper

Abstract

Quantitative analysis of genetically modified (GM) foods requires estimation of the amount of the transgenic event relative to an endogenous gene. Regulatory authorities in the European Union (EU) have defined the labelling threshold for GM food on the copy number ratio between the transgenic event and an endogenous gene. Real-time polymerase chain reaction (PCR) is currently being used for quantification of GM organisms (GMOs). Limitations in real-time PCR applications to detect very low number of DNA targets has led to new developments such as the digital PCR (dPCR) which allows accurate measurement of DNA copies without the need for a reference calibrator. In this paper, the amount of maize MON810 and hmg copies present in a DNA extract from seed powders certified for their mass content and for their copy number ratio was measured by dPCR. The ratio of these absolute copy numbers determined by dPCR was found to be identical to the ratios measured by real-time quantitative PCR (qPCR) using a plasmid DNA calibrator. These results indicate that both methods could be applied to determine the copy number ratio in MON810. The reported values were in agreement with estimations from a model elaborated to convert mass fractions into copy number fractions in MON810 varieties. This model was challenged on two MON810 varieties used for the production of MON810 certified reference materials (CRMs) which differ in the parental origin of the introduced GM trait. We conclude that dPCR has a high metrological quality and can be used for certifying GM CRMs in terms of DNA copy number ratio.

Keywords

PCR Maize Zea mays Genetically modified organism Biochips high-throughput screening Nucleic acids (DNA|RNA) 

Abbreviations

CRM

Certified reference material

DNA

Deoxyribonucleic acid

dPCR

Digital polymerase chain reaction

EC

European Commission

ERM

European reference material

EU

European Union

gDNA

Genomic DNA

GM

Genetically modified

GMO

Genetically modified organism

HG

Haploid genome

hmg

High-mobility-group protein A gene

MON810

MON810 gene

PCR

Polymerase chain reaction

qPCR

Quantitative polymerase chain reaction

SD

Standard deviation

RSD

Relative standard deviation

Notes

Acknowledgments

We thank S. Trapmann and H. Emons (IRMM) for their very useful review and constructive criticism of the manuscript.

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Copyright information

© Springer-Verlag 2009

Authors and Affiliations

  • Philippe Corbisier
    • 2
    Email author
  • Somanath Bhat
    • 1
  • Lina Partis
    • 1
  • Vicki Rui Dan Xie
    • 1
  • Kerry R. Emslie
    • 1
  1. 1.National Measurement InstituteLindfieldAustralia
  2. 2.European Commission, Joint Research CentreInstitute for Reference Materials and Measurements (IRMM)GeelBelgium

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