Abstract
We investigate the applicability of using digital PCR to estimate absolute limits of detection and quantitation associated with the validation of traditional real-time PCR methods for analysis of genetically modified (GM) ingredients. We also demonstrate the use of dynamic arrays as a precursor in selecting suitable sample dilution levels for accurate copy number assessment using the aforementioned digital PCR. Additionally, we further explore the relevance of digital PCR in accurately quantifying plasmid copy numbers associated with a commercially available Certified Reference Material. The use of digital PCR has the advantage of facilitating absolute single molecule detection, therefore negating the necessity for standards on a calibration curve, and reducing associated matrix effects. The approaches described in this paper enable pre-existing validated protocols to be re-examined, and estimates based on an alternative approach using digital PCR to be used in order to objectively characterise sensitivity limits.
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Acknowledgments
The work described in this paper was supported by the UK Department for Business, Innovation and Skills (BIS) as part of the “Government Chemist 2008–2011 Programme”. The authors gratefully acknowledge funding provided by the 6th EU Framework Programme for Research and Technological Development (FP6) project “GM and non-GM supply chains: their CO-Existence and TRAceability” (Contract number 007158).
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Burns, M.J., Burrell, A.M. & Foy, C.A. The applicability of digital PCR for the assessment of detection limits in GMO analysis. Eur Food Res Technol 231, 353–362 (2010). https://doi.org/10.1007/s00217-010-1277-8
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DOI: https://doi.org/10.1007/s00217-010-1277-8