Autoren:	Moser, Dirk A. Braga, Luca Raso, Andrea Zacchigna, Serena Giacca, Mauro Simon, Perikles
Titel:	Transgene detection by digital droplet PCR
Online-Publikationsdatum:	19-Aug-2022
Erscheinungsdatum:	2014
Sprache des Dokuments:	Englisch
Zusammenfassung/Abstract:	Somatic gene therapy is a promising tool for the treatment of severe diseases. Because of its abuse potential for performance enhancement in sports, the World Anti-Doping Agency (WADA) included the term 'gene doping' in the official list of banned substances and methods in 2004. Several nested PCR or qPCR-based strategies have been proposed that aim at detecting long-term presence of transgene in blood, but these strategies are hampered by technical limitations. We developed a digital droplet PCR (ddPCR) protocol for Insulin-Like Growth Factor 1 (IGF1) detection and demonstrated its applicability monitoring 6 mice injected into skeletal muscle with AAV9-IGF1 elements and 2 controls over a 33-day period. A duplex ddPCR protocol for simultaneous detection of Insulin-Like Growth Factor 1 (IGF1) and Erythropoietin (EPO) transgenic elements was created. A new DNA extraction procedure with target-orientated usage of restriction enzymes including on-column DNA-digestion was established. In vivo data revealed that IGF1 transgenic elements could be reliably detected for a 33-day period in DNA extracted from whole blood. In vitro data indicated feasibility of IGF1 and EPO detection by duplex ddPCR with high reliability and sensitivity. On-column DNA-digestion allowed for significantly improved target detection in downstream PCR-based approaches. As ddPCR provides absolute quantification, it ensures excellent day-to-day reproducibility. Therefore, we expect this technique to be used in diagnosing and monitoring of viral and bacterial infection, in detecting mutated DNA sequences as well as profiling for the presence of foreign genetic material in elite athletes in the future.
DDC-Sachgruppe:	796 Sport 796 Athletic and outdoor sports and games
Veröffentlichende Institution:	Johannes Gutenberg-Universität Mainz
Organisationseinheit:	FB 02 Sozialwiss., Medien u. Sport
Veröffentlichungsort:	Mainz
ROR:	https://ror.org/023b0x485
DOI:	http://doi.org/10.25358/openscience-7583
Version:	Published version
Publikationstyp:	Zeitschriftenaufsatz
Nutzungsrechte:	CC BY
Informationen zu den Nutzungsrechten:	https://creativecommons.org/licenses/by/4.0/
Zeitschrift:	PLoS one 9 11
Seitenzahl oder Artikelnummer:	e111781
Verlag:	PLoS
Verlagsort:	Lawrence, Kan.
Erscheinungsdatum:	2014
ISSN:	1932-6203
URL der Originalveröffentlichung:	http://dx.doi.org/10.1371/journal.pone.0111781
DOI der Originalveröffentlichung:	10.1371/journal.pone.0111781
Enthalten in den Sammlungen:	DFG-OA-Publizieren (2012 - 2017)