Gene doping control analysis of human erythropoietin transgene in equine plasma by PCR-liquid chromatography high resolution tandem mass spectrometry

Gene doping involves the misuse of gene materials to alter athlete’s performance which is banned at all times in both human and equine sports. Quantitative polymerase chain reaction (qPCR) assays have been used to control the misuse of transgenes in equine sports. Our laboratory recently developed and implemented duplex as well as multiplex qPCR assays for transgenes detection. As our continuous efforts in advancing gene doping control, in this work we have developed for the first time a sensitive and definitive PCR-liquid chromatography high resolution tandem mass spectrometry (PCR-LC-HRMS/MS) method for transgene detection, thus achieving an estimated limit of detection below 100 copies/mL for human erythropoietin (hEPO) transgene in equine plasma. The method involved magnetic-glass-particle-based extraction of DNA from equine plasma prior to PCR amplification with 2'-deoxyuridine 5'-triphosphate (dUTP), followed by treatments with uracil DNA glycosylase and hot piperidine for selective cleavage to give small oligonucleotide fragments. The resulting DNA fragments were then analysed by LC-HRMS/MS. The applicability of this method has been demonstrated by successful detection of hEPO transgene in blood samples collected from a gelding that had been administered with hEPO. This novel approach not only serves as an orthogonal method for transgene detection, but also paves the way to development of generic PCR-LC-HRMS/MS method for detection of multiple transgenes.