The advancements in gene therapy technologies raise significant concerns regarding their potential misuse for enhancing athletic performance. To fulfil the regulatory needs, gene doping detection methods have been developed. Specifically, PCR-based detection methods have been widely used due to its accuracy and reproducibility. However, robust confirmatory techniques for transgene detection in conventional doping control laboratories remain limited.
This research aims to develop a novel approach coupling PCR with liquid chromatography-high-resolution tandem mass spectrometry (PCR-LC-HRMS/MS) for the confirmatory analysis of transgenes in equine plasma. The method involved amplifying targeted transgenes using PCR in the presence of 2′-deoxyuridine 5′-triphosphate, followed by specific cleavages at the uracil bases using uracil DNA glycosylase and hot piperidine. This process generated unique DNA fragments corresponding to exon-exon junctions (EEJs) of transgenes, which were then detected and confirmed by LC-HRMS/MS.
Initially, a singleplex method targeting the human erythropoietin (hEPO) transgene was developed. It successfully detected and confirmed the presence of the hEPO transgene in plasma samples collected from a horse administered with the transgene, representing the first reported application of LC-HRMS/MS for transgene confirmation in equine plasma.
Recognizing the need for higher throughout and reliability, a multiplex method was subsequently developed. This method simultaneously targeted three potential doping transgenes: equine growth hormone 1, equine growth hormone-releasing hormone, and equine interleukin 10 (eIL10), alongside an artificial internal control to mitigate false negatives. Specific EEJ fragments for each target were designed, generated via the uracil cleavage strategy, and detected by LC-HRMS/MS. The multiplex method was validated using equine plasma and successfully applied to detect the eIL10 transgene post-administration.
These PCR-LC-HRMS/MS methods provide a novel confirmatory platform for gene doping control. The use of conventional LC-HRMS/MS makes this a practical and adaptable solution for anti-doping laboratories to effectively combat the emerging threat of gene doping in equine sports.