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Development of event-specific qPCR detection methods for genetically modified alfalfa events J101, J163 and KK179

Genetically modified alfalfa is authorized for cultivation in several countries since 2005. On the other hand, cultivation in or export to the European Union is not allowed and thus neither certified reference material nor official event-specific detection methods are available. Therefore, based on...

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Detalles Bibliográficos
Autores principales: Guertler, Patrick, Grohmann, Lutz, Naumann, Heike, Pavlovic, Melanie, Busch, Ulrich
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Elsevier 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6446038/
https://www.ncbi.nlm.nih.gov/pubmed/30984566
http://dx.doi.org/10.1016/j.bdq.2018.12.001
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author Guertler, Patrick
Grohmann, Lutz
Naumann, Heike
Pavlovic, Melanie
Busch, Ulrich
author_facet Guertler, Patrick
Grohmann, Lutz
Naumann, Heike
Pavlovic, Melanie
Busch, Ulrich
author_sort Guertler, Patrick
collection PubMed
description Genetically modified alfalfa is authorized for cultivation in several countries since 2005. On the other hand, cultivation in or export to the European Union is not allowed and thus neither certified reference material nor official event-specific detection methods are available. Therefore, based on patent sequence information, event-specific real-time PCR detection methods targeting the junction sequence of the alfalfa genome and the transgenic insert of the respective events J101, J163 and KK179 were developed. Newly developed plasmids were used as reference material for assay optimization and in-house validation. Plasmid standards were quantified using digital droplet PCR and LOD95%, PCR efficiency, robustness and specificity of the assays were determined using real-time PCR. A LOD95% of 10 copies per PCR reaction was observed and PCR efficiencies of 95–97 % were achieved. Different real-time PCR instruments and PCR conditions were applied to test for robustness of the assays using DNA at a concentration of 30 copies per μL for each gm alfalfa event. All replicates were positive independent of the instrument or the PCR condition. DNA from certified reference material of different genetically modified crops as well as reference materials of the three events was used to experimentally test for specificity. No unspecific amplification signal was observed for any of the assays. Validation results were in line with the “Minimum Performance Requirements for Analytical Methods of GMO Testing” of the European Network of GMO Laboratories. Furthermore, an inter-laboratory comparison study was conducted to show the transferability and applicability of the methods and to verify the assay performance parameters.
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spelling pubmed-64460382019-04-12 Development of event-specific qPCR detection methods for genetically modified alfalfa events J101, J163 and KK179 Guertler, Patrick Grohmann, Lutz Naumann, Heike Pavlovic, Melanie Busch, Ulrich Biomol Detect Quantif Special Issue on Liquid Biopsy & Biomarkers, Edited by Dr Michael Pfaffl Genetically modified alfalfa is authorized for cultivation in several countries since 2005. On the other hand, cultivation in or export to the European Union is not allowed and thus neither certified reference material nor official event-specific detection methods are available. Therefore, based on patent sequence information, event-specific real-time PCR detection methods targeting the junction sequence of the alfalfa genome and the transgenic insert of the respective events J101, J163 and KK179 were developed. Newly developed plasmids were used as reference material for assay optimization and in-house validation. Plasmid standards were quantified using digital droplet PCR and LOD95%, PCR efficiency, robustness and specificity of the assays were determined using real-time PCR. A LOD95% of 10 copies per PCR reaction was observed and PCR efficiencies of 95–97 % were achieved. Different real-time PCR instruments and PCR conditions were applied to test for robustness of the assays using DNA at a concentration of 30 copies per μL for each gm alfalfa event. All replicates were positive independent of the instrument or the PCR condition. DNA from certified reference material of different genetically modified crops as well as reference materials of the three events was used to experimentally test for specificity. No unspecific amplification signal was observed for any of the assays. Validation results were in line with the “Minimum Performance Requirements for Analytical Methods of GMO Testing” of the European Network of GMO Laboratories. Furthermore, an inter-laboratory comparison study was conducted to show the transferability and applicability of the methods and to verify the assay performance parameters. Elsevier 2019-02-11 /pmc/articles/PMC6446038/ /pubmed/30984566 http://dx.doi.org/10.1016/j.bdq.2018.12.001 Text en © 2018 The Authors http://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
spellingShingle Special Issue on Liquid Biopsy & Biomarkers, Edited by Dr Michael Pfaffl
Guertler, Patrick
Grohmann, Lutz
Naumann, Heike
Pavlovic, Melanie
Busch, Ulrich
Development of event-specific qPCR detection methods for genetically modified alfalfa events J101, J163 and KK179
title Development of event-specific qPCR detection methods for genetically modified alfalfa events J101, J163 and KK179
title_full Development of event-specific qPCR detection methods for genetically modified alfalfa events J101, J163 and KK179
title_fullStr Development of event-specific qPCR detection methods for genetically modified alfalfa events J101, J163 and KK179
title_full_unstemmed Development of event-specific qPCR detection methods for genetically modified alfalfa events J101, J163 and KK179
title_short Development of event-specific qPCR detection methods for genetically modified alfalfa events J101, J163 and KK179
title_sort development of event-specific qpcr detection methods for genetically modified alfalfa events j101, j163 and kk179
topic Special Issue on Liquid Biopsy & Biomarkers, Edited by Dr Michael Pfaffl
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6446038/
https://www.ncbi.nlm.nih.gov/pubmed/30984566
http://dx.doi.org/10.1016/j.bdq.2018.12.001
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