Establishment of two quantitative nested qPCR assays targeting the human EPO transgene

Autoren

EWI Neuberger
I Perez
C Le Guiner
D Moser
T Ehlert
M Allais
P Moullier
P Simon
RO Snyder

Autoren-URL

https://www.webofscience.com/api/gateway?GWVersion=2&SrcApp=fis-test-1&SrcAuth=WosAPI&KeyUT=WOS:000374125400002&DestLinkType=FullRecord&DestApp=WOS_CPL

DOI

10.1038/gt.2016.2

eISSN

1476-5462

Externe Identifier

Clarivate Analytics Document Solution ID: DJ3SE
PubMed Identifier: 26752352

ISSN

0969-7128

Ausgabe der Veröffentlichung

4

Zeitschrift

GENE THERAPY

Paginierung

330 - 339

Datum der Veröffentlichung

2016

Status

Published

Titel

Establishment of two quantitative nested qPCR assays targeting the human EPO transgene

Sub types

Article

Ausgabe der Zeitschrift

23

Datenquelle: Web of Science (Lite)

Autoren

EWI Neuberger
I Perez
C Le Guiner
D Moser
T Ehlert
M Allais
P Moullier
P Simon
RO Snyder

DOI

10.1038/gt.2016.2

eISSN

1476-5462

ISSN

0969-7128

Ausgabe der Veröffentlichung

4

Zeitschrift

Gene Therapy

Sprache

en

Online publication date

2016

Paginierung

330 - 339

Datum der Veröffentlichung

2016

Status

Published

Herausgeber

Springer Science and Business Media LLC

Herausgeber URL

http://dx.doi.org/10.1038/gt.2016.2

Datum der Datenerfassung

2022

Titel

Establishment of two quantitative nested qPCR assays targeting the human EPO transgene

Ausgabe der Zeitschrift

23

Datenquelle: Crossref

Abstract

For ethical and safety reasons it is critical to develop easily implemented assays with high sensitivity and specificity for gene doping surveillance. Two nested quantitative real-time PCR (qPCR) assays were developed that target the human EPO (hEPO) cDNA sequence in a circular form, representative of recombinant adeno-associated viral (rAAV) vector genomes found in vivo. Through an interlaboratory evaluation, the assays were validated and utilized in an in vitro blinded study. These assays are specific and extremely sensitive with a limit of detection (LOD) of 1 copy of circular plasmid DNA and a limit of quantification (LOQ) of 10 to 20 copies in the presence of 500 ng of human genomic DNA (hgDNA) extracted from WBCs. Additionally, using the two nested qPCR assays in a non-human primate study, where macaques were injected intramuscularly with a rAAV8 vector harboring a promoterless hEPO cDNA sequence, the viral vector was detected 8 to 14 weeks post-injection in macaque WBCs. The high sensitivity of the nested qPCR approach along with the capability of quantifying target DNA, make this approach a reliable tool for gene doping surveillance and the monitoring of exogenous DNA sequences.

Addresses

Faculty of Social Science, Media and Sport, Department of Sports Medicine, Johannes Gutenberg-University Mainz, Mainz, Germany.

Autoren

EWI Neuberger
I Perez
C Le Guiner
D Moser
T Ehlert
M Allais
P Moullier
P Simon
RO Snyder

DOI

10.1038/gt.2016.2

eISSN

1476-5462

Externe Identifier

PubMed Identifier: 26752352

Open access

false

ISSN

0969-7128

Ausgabe der Veröffentlichung

4

Zeitschrift

Gene therapy

Schlüsselwörter

Animals
Macaca
Macaca fascicularis
Humans
Erythropoietin
DNA
Sensitivity and Specificity
Polymerase Chain Reaction
Transgenes
Genetic Vectors
Plasmids
Doping in Sports
Male
Real-Time Polymerase Chain Reaction

Sprache

eng

Medium

Print-Electronic

Online publication date

2016

Paginierung

330 - 339

Datum der Veröffentlichung

2016

Status

Published

Datum der Datenerfassung

2016

Titel

Establishment of two quantitative nested qPCR assays targeting the human EPO transgene.

Sub types

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

Ausgabe der Zeitschrift

23

Datenquelle: Europe PubMed Central

Abstract

For ethical and safety reasons it is critical to develop easily implemented assays with high sensitivity and specificity for gene doping surveillance. Two nested quantitative real-time PCR (qPCR) assays were developed that target the human EPO (hEPO) cDNA sequence in a circular form, representative of recombinant adeno-associated viral (rAAV) vector genomes found in vivo. Through an interlaboratory evaluation, the assays were validated and utilized in an in vitro blinded study. These assays are specific and extremely sensitive with a limit of detection (LOD) of 1 copy of circular plasmid DNA and a limit of quantification (LOQ) of 10 to 20 copies in the presence of 500 ng of human genomic DNA (hgDNA) extracted from WBCs. Additionally, using the two nested qPCR assays in a non-human primate study, where macaques were injected intramuscularly with a rAAV8 vector harboring a promoterless hEPO cDNA sequence, the viral vector was detected 8 to 14 weeks post-injection in macaque WBCs. The high sensitivity of the nested qPCR approach along with the capability of quantifying target DNA, make this approach a reliable tool for gene doping surveillance and the monitoring of exogenous DNA sequences.

Date of acceptance

2015

Autoren

EWI Neuberger
I Perez
C Le Guiner
D Moser
T Ehlert
M Allais
P Moullier
P Simon
RO Snyder

Autoren-URL

https://www.ncbi.nlm.nih.gov/pubmed/26752352

DOI

10.1038/gt.2016.2

eISSN

1476-5462

Ausgabe der Veröffentlichung

4

Zeitschrift

Gene Ther

Schlüsselwörter

Animals
DNA
Doping in Sports
Erythropoietin
Genetic Vectors
Humans
Macaca
Macaca fascicularis
Male
Plasmids
Polymerase Chain Reaction
Real-Time Polymerase Chain Reaction
Sensitivity and Specificity
Transgenes

Sprache

eng

Country

England

Paginierung

330 - 339

PII

gt20162

Datum der Veröffentlichung

2016

Status

Published

Datum, an dem der Datensatz öffentlich gemacht wurde

2017

Titel

Establishment of two quantitative nested qPCR assays targeting the human EPO transgene.

Sub types

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

Ausgabe der Zeitschrift

23

Datenquelle: PubMed

Establishment of two quantitative nested qPCR assays targeting the human EPO transgene

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