This article presents an optimized protocol for gene doping detection. Starting from one spot DBS (20 μL), concentrated DNA was obtained and real-time PCR parameters were adjusted for increased detection. Robust detection of 5000 copies/mL of transgene was validated for polymeric device TASSO M-20.

ABSTRACT

For the past couple of years, black market products have appeared and were confirmed to contain genetic products coding for human erythropoietin (EPO). While being prohibited by the World Anti-Doping Agency (WADA), they could be used to produce endogenously more EPO hormone and hence increase performance. In a previous work, we demonstrated the potential of 20-μL dried blood spots (DBS) to detect the presence of EPO transgene in human blood down to 250 copies (12,500 copies/mL), despite lower sensitivity (30-fold) than in 1-mL fresh blood. As the use of DBS as a collection matrix for antidoping is going to expand in the near future, our aim was to develop and validate a new protocol to improve the sensitivity of gene doping detection from DBS. Three DBS devices were evaluated: polymeric Tasso-M20 (TASSO Inc.) and Mitra (Neoteryx), and cellulosic Protein Saver 903 (Whatman). The best results were achieved with polymeric DBS, and a full validation was performed for the detection of the EPO transgene using Tasso M-20 DBS; 1500 copies/mL were detected in 50% of cases and robust detection was obtained at 5000 copies/mL (100 copies transgene in 20-μL DBS) with the four spots of the Tasso device tested over several weeks. The results confirm that polymeric DBS can be used as an alternative to fresh blood for gene doping detection with high sensitivity simplifying also potential reanalysis in the future.