The practice of doping threatens fair competition in sports. With the very recent reports on successful gene therapies for several diseases, the likelihood for abuse of gene transfer techniques in elite sports is rapidly increasing. It is therefore important to develop valid detection techniques, indicating the transfer of DNA (tDNA), with ultimate sensitivity and specificity. To date, three slightly different procedures have been reported to reliably detect tDNA with high sensitivity. Two are using a real-time PCR based approach and one is using a primer-internal, intron-spanning PCR approach (spiPCR). The specificity and sensitivity of these techniques, however, is still a matter of debate.

Based on spiPCR, here we present a novel one-tube nested PCR approach that minimizes the chances for cross-contamination and shows increased sensitivity compared to non-nested PCR techniques. To further reduce the occurrence of false-positives based on cross-contamination, a multi-functional 19bp extended erythropoietin standard (EPO) was cloned which can be easily differentiated from EPO tDNA and can be used as an internal or external positive control also in real-time PCR based applications.

We found that one-tube nested PCR is superior in terms of sensitivity and specificity compared to conventional PCR and shows similar sensitivity compared to real-time based pcr assays. However, it didn't reach sensitivity of spiPCR but as it is less laborious and expensive and much faster it could be used as a pre-screening tool in the future.

We hope that the sensitivity of the currently developed molecular biological detection techniques in combination with their ability to allow long-term tDNA detection will deter athletes from using doping techniques based on gene transfer.