| Popis: |
Gene doping, the misuse of gene therapy for performance enhancement, is predicted to be the next phase of doping faced by horseracing. Since 2003, the World Anti-Doping Agency has included gene doping on the list of banned practices in human sports and has several expert teams developing detection methods. The threat of gene doping to the integrity of horseracing and welfare of horses has been discussed by members of The International Federation of Horseracing Authorities and others in the anti-doping community for over ten years. In this thesis, a method is developed for direct detection of transfer DNA (T-DNA) in equine blood samples. The method uses real-time polymerase chain reaction (RT-qPCR) to target unique ‘foreign’ sequences in T-DNA. Ten RT-qPCR assays were developed targeting T-DNA for five equine genes predicted to be high priority equine gene doping targets. Based on standard practice in horseracing drug analysis, the gene doping test consists of two stages, screening and confirmation. To increase sample throughput in the screening stage, assays for four gene targets were optimised and validated as a multiplex suite. The assays were optimised and validated using two in vitro systems that mimic positive doping samples. This work develops the entire doping detection test from sample collection to confirmation of a positive sample. The developed method detected as few as 300 copies of T-DNA per millilitre of blood, comparable to tests developed for human gene doping detection. In a world-first for gene doping surveillance, the developed gene doping test was used to screen real samples for Racing NSW as part of out of competition surveillance prior to the Sydney Autumn Racing Carnival 2018. The results showed that gene doping detection is feasible using the method developed in this project. The test developed here will be directly applicable to all breeds of horses involved in both racing and other athletic competition. |
