Transgene detection by digital droplet PCR

Autoren

Dirk A Moser
Luca Braga
Andrea Raso
Serena Zacchigna
Mauro Giacca
Perikles Simon

Autoren-URL

https://www.webofscience.com/api/gateway?GWVersion=2&SrcApp=fis-test-1&SrcAuth=WosAPI&KeyUT=WOS:000344402600062&DestLinkType=FullRecord&DestApp=WOS_CPL

DOI

10.1371/journal.pone.0111781

Externe Identifier

Clarivate Analytics Document Solution ID: AS6XM
PubMed Identifier: 25375130

ISSN

1932-6203

Ausgabe der Veröffentlichung

11

Zeitschrift

PLOS ONE

Artikelnummer

ARTN e111781

Datum der Veröffentlichung

2014

Status

Published

Titel

Transgene Detection by Digital Droplet PCR

Sub types

Article

Ausgabe der Zeitschrift

9

Datenquelle: Web of Science (Lite)

Autoren

Dirk A Moser
Luca Braga
Andrea Raso
Serena Zacchigna
Mauro Giacca
Perikles Simon

DOI

10.1371/journal.pone.0111781

Editoren

Domenico Coppola

eISSN

1932-6203

Ausgabe der Veröffentlichung

11

Zeitschrift

PLoS ONE

Sprache

en

Online publication date

2014

Paginierung

e111781 - e111781

Status

Published online

Herausgeber

Public Library of Science (PLoS)

Herausgeber URL

http://dx.doi.org/10.1371/journal.pone.0111781

Datum der Datenerfassung

2020

Titel

Transgene Detection by Digital Droplet PCR

Ausgabe der Zeitschrift

9

Datenquelle: Crossref

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.

Addresses

Faculty of Psychology, Genetic Psychology, Ruhr-University-Bochum, Bochum, Germany; Department of Sports Medicine, Disease Prevention and Rehabilitation, Johannes Gutenberg-University Mainz, Mainz, Germany.

Autoren

Dirk A Moser
Luca Braga
Andrea Raso
Serena Zacchigna
Mauro Giacca
Perikles Simon

DOI

10.1371/journal.pone.0111781

eISSN

1932-6203

Externe Identifier

PubMed Identifier: 25375130
PubMed Central ID: PMC4222945

Open access

true

ISSN

1932-6203

Ausgabe der Veröffentlichung

11

Zeitschrift

PloS one

Schlüsselwörter

Animals
Humans
Mice
Dependovirus
Insulin-Like Growth Factor I
Erythropoietin
Injections, Intramuscular
Reproducibility of Results
Polymerase Chain Reaction
Transgenes
Genetic Vectors
In Vitro Techniques

Sprache

eng

Medium

Electronic-eCollection

Online publication date

2014

Open access status

Open Access

Paginierung

e111781

Datum der Veröffentlichung

2014

Status

Published

Publisher licence

CC BY

Datum der Datenerfassung

2014

Titel

Transgene detection by digital droplet PCR.

Sub types

Research Support, Non-U.S. Gov't
research-article
Journal Article

Ausgabe der Zeitschrift

9

Files

https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0111781&type=printable https://europepmc.org/articles/PMC4222945?pdf=render

Datenquelle: Europe PubMed Central

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.

Date of acceptance

2014

Autoren

Dirk A Moser
Luca Braga
Andrea Raso
Serena Zacchigna
Mauro Giacca
Perikles Simon

Autoren-URL

https://www.ncbi.nlm.nih.gov/pubmed/25375130

DOI

10.1371/journal.pone.0111781

eISSN

1932-6203

Externe Identifier

PubMed Central ID: PMC4222945

Ausgabe der Veröffentlichung

11

Zeitschrift

PLoS One

Schlüsselwörter

Animals
Dependovirus
Erythropoietin
Genetic Vectors
Humans
In Vitro Techniques
Injections, Intramuscular
Insulin-Like Growth Factor I
Mice
Polymerase Chain Reaction
Reproducibility of Results
Transgenes

Sprache

eng

Country

United States

Paginierung

e111781

PII

PONE-D-14-19024

Datum der Veröffentlichung

2014

Status

Published online

Datum, an dem der Datensatz öffentlich gemacht wurde

2015

Titel

Transgene detection by digital droplet PCR.

Sub types

Journal Article
Research Support, Non-U.S. Gov't

Ausgabe der Zeitschrift

9

Datenquelle: PubMed

Author's licence

CC-BY

Autoren

Dirk A Moser
Luca Braga
Andrea Raso
Serena Zacchigna
Mauro Giacca
Perikles Simon

Hosting institution

Universitätsbibliothek Mainz

Sammlungen

DFG-OA-Publizieren (2012 - 2017)

Resource version

Published version

DOI

10.1371/journal.pone.0111781

Funding acknowledgements

DFG, Open Access-Publizieren Universität Mainz / Universitätsmedizin

File(s) embargoed

false

Open access

true

ISSN

1932-6203

Ausgabe der Veröffentlichung

11

Zeitschrift

PLoS one

Schlüsselwörter

796 Sport
796 Athletic and outdoor sports and games

Sprache

eng

Open access status

Open Access

Paginierung

e111781

Datum der Veröffentlichung

2014

Public URL

https://openscience.ub.uni-mainz.de/handle/20.500.12030/7597

Herausgeber

PLoS

Herausgeber URL

http://dx.doi.org/10.1371/journal.pone.0111781

Datum der Datenerfassung

2022

Datum, an dem der Datensatz öffentlich gemacht wurde

2022

Zugang

Public

Titel

Transgene detection by digital droplet PCR

Ausgabe der Zeitschrift

9

Files

transgene_detection_by_digita-20220817172133134.pdf

Datenquelle: OPENSCIENCE.UB

Files

Files

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