Inter-laboratory analysis of selected genetically modified plant reference materials with digital PCR

Digital PCR (dPCR), as a new technology in the field of genetically modified (GM) organism (GMO) testing, enables determination of absolute target copy numbers. The purpose of our study was to test the transferability of methods designed for quantitative PCR (qPCR) to dPCR and to carry out an inter-...

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Autores principales: Dobnik, David, Demšar, Tina, Huber, Ingrid, Gerdes, Lars, Broeders, Sylvia, Roosens, Nancy, Debode, Frederic, Berben, Gilbert, Žel, Jana
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Springer Berlin Heidelberg 2017
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5748423/
https://www.ncbi.nlm.nih.gov/pubmed/29071363
http://dx.doi.org/10.1007/s00216-017-0711-1
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author Dobnik, David
Demšar, Tina
Huber, Ingrid
Gerdes, Lars
Broeders, Sylvia
Roosens, Nancy
Debode, Frederic
Berben, Gilbert
Žel, Jana
author_facet Dobnik, David
Demšar, Tina
Huber, Ingrid
Gerdes, Lars
Broeders, Sylvia
Roosens, Nancy
Debode, Frederic
Berben, Gilbert
Žel, Jana
author_sort Dobnik, David
collection PubMed
description Digital PCR (dPCR), as a new technology in the field of genetically modified (GM) organism (GMO) testing, enables determination of absolute target copy numbers. The purpose of our study was to test the transferability of methods designed for quantitative PCR (qPCR) to dPCR and to carry out an inter-laboratory comparison of the performance of two different dPCR platforms when determining the absolute GM copy numbers and GM copy number ratio in reference materials certified for GM content in mass fraction. Overall results in terms of measured GM% were within acceptable variation limits for both tested dPCR systems. However, the determined absolute copy numbers for individual genes or events showed higher variability between laboratories in one third of the cases, most possibly due to variability in the technical work, droplet size variability, and analysis of the raw data. GMO quantification with dPCR and qPCR was comparable. As methods originally designed for qPCR performed well in dPCR systems, already validated qPCR assays can most generally be used for dPCR technology with the purpose of GMO detection. [Figure: see text]
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spelling pubmed-57484232018-01-19 Inter-laboratory analysis of selected genetically modified plant reference materials with digital PCR Dobnik, David Demšar, Tina Huber, Ingrid Gerdes, Lars Broeders, Sylvia Roosens, Nancy Debode, Frederic Berben, Gilbert Žel, Jana Anal Bioanal Chem Research Paper Digital PCR (dPCR), as a new technology in the field of genetically modified (GM) organism (GMO) testing, enables determination of absolute target copy numbers. The purpose of our study was to test the transferability of methods designed for quantitative PCR (qPCR) to dPCR and to carry out an inter-laboratory comparison of the performance of two different dPCR platforms when determining the absolute GM copy numbers and GM copy number ratio in reference materials certified for GM content in mass fraction. Overall results in terms of measured GM% were within acceptable variation limits for both tested dPCR systems. However, the determined absolute copy numbers for individual genes or events showed higher variability between laboratories in one third of the cases, most possibly due to variability in the technical work, droplet size variability, and analysis of the raw data. GMO quantification with dPCR and qPCR was comparable. As methods originally designed for qPCR performed well in dPCR systems, already validated qPCR assays can most generally be used for dPCR technology with the purpose of GMO detection. [Figure: see text] Springer Berlin Heidelberg 2017-10-25 2018 /pmc/articles/PMC5748423/ /pubmed/29071363 http://dx.doi.org/10.1007/s00216-017-0711-1 Text en © The Author(s) 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.
spellingShingle Research Paper
Dobnik, David
Demšar, Tina
Huber, Ingrid
Gerdes, Lars
Broeders, Sylvia
Roosens, Nancy
Debode, Frederic
Berben, Gilbert
Žel, Jana
Inter-laboratory analysis of selected genetically modified plant reference materials with digital PCR
title Inter-laboratory analysis of selected genetically modified plant reference materials with digital PCR
title_full Inter-laboratory analysis of selected genetically modified plant reference materials with digital PCR
title_fullStr Inter-laboratory analysis of selected genetically modified plant reference materials with digital PCR
title_full_unstemmed Inter-laboratory analysis of selected genetically modified plant reference materials with digital PCR
title_short Inter-laboratory analysis of selected genetically modified plant reference materials with digital PCR
title_sort inter-laboratory analysis of selected genetically modified plant reference materials with digital pcr
topic Research Paper
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5748423/
https://www.ncbi.nlm.nih.gov/pubmed/29071363
http://dx.doi.org/10.1007/s00216-017-0711-1
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