Detection using chamber digital PCR with a DNA extraction-free method for gene-doping control

Gene doping, which entails the administration of transgenes, poses a serious threat to the integrity of equine sports and also raises both ethical and regulatory concerns. Current methods used for the detection of such doping often necessitate the extraction of DNA from plasma, which can be time-consuming and labour-intensive. To overcome this limitation, we developed a direct chamber digital PCR (cdPCR) method that enables transgene detection in equine plasma without the need for DNA purification. Using the equine erythropoietin (EPO) transgene as a model, we validated the assay by analysing plasma samples spiked at 10-1000 copies/µL. Samples were pre-treated with Lysis Buffer S Ver.2 and used directly as templates for cdPCR, employing a hydrolysis probe targeting an exon-exon junction of the transgene. This method required only 0.55 µL of plasma per 10 µL reaction and enabled us to achieve consistent detection down to 1000 copies/µL with high reproducibility and low background interference. The assay also proved effective in detecting the EPO transgene in plasma collected from a Thoroughbred horse after intramuscular plasmid administration. Compared with conventional PCR-based methods, this protocol substantially reduces sample handling and processing times, without any appreciable decrement in sensitivity. Our findings indicate the potential utility of direct cdPCR as a practical tool for gene doping surveillance in equine sports. Its simplified workflow and minimal sample requirement also suggest broader applications in forensic biotechnology, for which rapid and reliable detection of genetic material is essential.