title: Establishment of two quantitative nested qPCR assays targeting the human EPO transgene.
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.
authors.affiliation: Faculty of Social Science, Media and Sport, Department of Sports Medicine, Johannes Gutenberg-University Mainz, Mainz, Germany.
authors.forename: E W I
authors.initials: EW
authors.lastname: Neuberger
authors.affiliation: Department of Molecular Genetics and Microbiology, University of Florida, College of Medicine, Gainesville, FL, USA.
authors.forename: I
authors.initials: I
authors.lastname: Perez
authors.affiliation: INSERM UMR1089, Atlantic Gene Therapies, CHU de Nantes, Université de Nantes, Nantes, France.|Genethon, Evry, France.
authors.forename: C
authors.initials: C
authors.lastname: Le Guiner
authors.affiliation: Faculty of Social Science, Media and Sport, Department of Sports Medicine, Johannes Gutenberg-University Mainz, Mainz, Germany.|Faculty of Psychology, Genetic Psychology, Ruhr-University-Bochum, Bochum, Germany.
authors.forename: D
authors.initials: D
authors.lastname: Moser
authors.affiliation: Faculty of Social Science, Media and Sport, Department of Sports Medicine, Johannes Gutenberg-University Mainz, Mainz, Germany.
authors.forename: T
authors.initials: T
authors.lastname: Ehlert
authors.affiliation: INSERM UMR1089, Atlantic Gene Therapies, CHU de Nantes, Université de Nantes, Nantes, France.
authors.forename: M
authors.initials: M
authors.lastname: Allais
authors.affiliation: Department of Molecular Genetics and Microbiology, University of Florida, College of Medicine, Gainesville, FL, USA.|INSERM UMR1089, Atlantic Gene Therapies, CHU de Nantes, Université de Nantes, Nantes, France.
authors.forename: P
authors.initials: P
authors.lastname: Moullier
authors.affiliation: Faculty of Social Science, Media and Sport, Department of Sports Medicine, Johannes Gutenberg-University Mainz, Mainz, Germany.
authors.forename: P
authors.initials: P
authors.lastname: Simon
authors.affiliation: Department of Molecular Genetics and Microbiology, University of Florida, College of Medicine, Gainesville, FL, USA.|INSERM UMR1089, Atlantic Gene Therapies, CHU de Nantes, Université de Nantes, Nantes, France.|Center of Excellence for Regenerative Health Biotechnology, University of Florida, Alachua, FL, USA.
authors.forename: R O
authors.initials: RO
authors.lastname: Snyder
mesh_terms: D000818:Animals; D004247:DNA / Q000097:blood / Q000235:genetics / Q000302:isolation & purification; D004300:Doping in Sports / Q000517:prevention & control; D004921:Erythropoietin / Q000032:analysis / Q000097:blood / Q000235:genetics*; D005822:Genetic Vectors; D006801:Humans; D008251:Macaca; D008252:Macaca fascicularis; D008297:Male; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D060888:Real-Time Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D019076:Transgenes
chemical_list: C554270:EPO protein, human; D004921:Erythropoietin; D004247:DNA