Authors:	Moser, Dirk A. Braga, Luca Raso, Andrea Zacchigna, Serena Giacca, Mauro Simon, Perikles
Title:	Transgene detection by digital droplet PCR
Online publication date:	19-Aug-2022
Year of first publication:	2014
Language:	english
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:	796 Sport 796 Athletic and outdoor sports and games
Institution:	Johannes Gutenberg-Universität Mainz
Department:	FB 02 Sozialwiss., Medien u. Sport
Place:	Mainz
ROR:	https://ror.org/023b0x485
DOI:	http://doi.org/10.25358/openscience-7583
Version:	Published version
Publication type:	Zeitschriftenaufsatz
License:	CC BY
Information on rights of use:	https://creativecommons.org/licenses/by/4.0/
Journal:	PLoS one 9 11
Pages or article number:	e111781
Publisher:	PLoS
Publisher place:	Lawrence, Kan.
Issue date:	2014
ISSN:	1932-6203
Publisher URL:	http://dx.doi.org/10.1371/journal.pone.0111781
Publisher DOI:	10.1371/journal.pone.0111781
Appears in collections:	DFG-OA-Publizieren (2012 - 2017)