Towards a Comprehensive Screening Approach for Detecting Gene Doping in Racehorses and Sport Horses by Targeting Common and Intrinsic Plasmid Sequences

The evolution of biotechnology and gene therapy has unfortunately facilitated the emergence of gene doping. Those misused applications are prohibited in equestrian sports (International Equestrian Federation (FEI)) and in horseracing (International Federation of Horseracing Authorities [IFHA]), reflecting major concerns regarding horse welfare and the integrity of competitions. Among the biotechnological tools that could be misused, eukaryotic expression plasmids represent a particular threat due to their ease of production, customisable design and ability to carry a wide range of performance transgenes. Current detection methods are mainly based on the identification of these transgenes, but if the sequence is unknown or modified, administration may not be detected. The aim of this work was to overcome this limitation by developing an untargeted transgene screening approach in equine plasma. A database of 349 mammalian expression plasmids was compiled, allowing identification of four conserved targets: the ampicillin resistance gene (AmpR), the cytomegalovirus promoter (pCMV) and two unannotated sequences between the features, which together covered 100% of the plasmids database. A set of PCR hydrolysis probe assays targeting these sequences has been developed and exhibited excellent efficiency, linearity and robustness. The method was able to discriminate, in plasma, 100% of suspicious samples from background noise down to 1200 copies/mL. Following plasmid administration to a horse, all four targets remained detectable up to 48 h using qPCR and dPCR, demonstrating the proof of concept. Overall, this work presented a sensitive, reliable and transgene‐independent method that strengthens plasmid detection and reinforces gene doping control in equine athletes.