Gene doping, which includes the non-therapeutic use of genes or genetic elements that have the capacity to enhance athletic performance, is prohibited in horseracing and equestrian sports. One such method is the use of gene transfer via a viral or plasmid vector to provide additional copies of genes involved in pathways such as muscle development or injury repair. The transgene payloads can be detected by a variety of techniques, many of which are qPCR-based.
We have taken a dual approach to screening and confirmatory analysis of transgenes. The screening pipeline employs a novel next-generation sequencing method which allows multiple assays to be performed in one PCR reaction, and up to 384 samples per sequencing run. Subsequent confirmatory analysis is then performed to established AORC guidelines via real time qPCR, with an additional step of sequencing to verify the amplicon identity. To verify both techniques for detection in a real doping scenario, an administration study of a bespoke rAAV vector was conducted to evaluate multiple matrices and technologies.
The approaches to validation for both the screening and confirmatory pipelines are discussed in detail, including mock confirmatory analysis of blind samples and double-blind controls for screening. ISO17025 accreditation has now been granted for both screening and confirmatory workflows. Consequently, routine screening is now in place in the UK, with additional pilot studies using the presented methodology being conducted by multiple jurisdictions around the world, including those within Australia, Ireland, the Middle East, and North America.