The advancement of genetic technology offers great promise for treating genetic diseases. However, this also raises the potential for them to be misused in sports to alter performance in human and animal athletes, termed as gene doping. It is imperative to develop a method to detect gene doping to deter its use, not only to uphold fairness in sports but protect the welfare of athletes. Detection methods of gene doping by gene transfer have been primarily PCR-based methods. Currently, equine gene doping detection has been primarily focused on equine targets.
At present, our laboratory screens for over 30 equine transgenes in one PCR reaction using massively parallel sequencing. The availability of human-sequence transgenes, however, as research materials and beyond, with purported availability on the internet, highlights the need to expand into other areas. To include human targets in an additional assay pool, primers for a selection of known performance human transgenes were designed, tested for specificity using cell-free and genomic DNA controls from horse and humans. Successful primers were used to create multiplexing panels to target 10 human-sequence transgenes per reaction via MPS. The effect of primer combinations within pools to the amplification of primer dimers was also investigated.
One of the panels was tested on 100 out-of-competition equine plasma samples and no amplification of the human transgenes was observed. Human transgenes spiked into plasma could be detected down to 250 copies per mL of plasma. The addition of human gene targets within a wider or additional pool will provide a vital tool against the growing portfolio of possible gene doping entry points.