Abstract
Indiscriminate genetic manipulation to improve athletic ability is a major threat to human sports and the horseracing industry, in which methods involving gene-doping, such as transgenesis, should be prohibited to ensure fairness. Therefore, development of methods to detect indiscriminate genetic manipulation are urgently needed. Here, we developed a highly sensitive method to detect horse erythropoietin (EPO) transgenes using droplet digital PCR (ddPCR). We designed two TaqMan probe/primer sets, and the EPO transgene was cloned into a plasmid for use as a model. We extracted the spiked EPO transgene from horse plasma and urine via magnetic beads, followed by ddPCR amplification for absolute quantification and transgene detection. The results indicated high recovery rates (at least ~60% and ~40% in plasma and urine, respectively), suggesting successful detection of the spiked transgene at concentrations of >130 and 200 copies/mL of plasma and urine, respectively. Additionally, successful detection was achieved following intramuscular injection of 20 mg of the EPO transgene. This represents the first study demonstrating a method for detecting the EPO transgene in horse plasma and urine, with our results demonstrating its efficacy for promoting the control of gene-doping in the horseracing industry.
Highlights
Horseracing began in Britain in the early 18th century and currently occurs worldwide
At day 2, the correlation coefficients were 0.9959 and 0.9986 for SET1 and SET2 using SD4.0 through SD8.5, with amplification efficiencies of 1.0225 and 0.9889, respectively (Figure 1b). These results indicated that SET1 and SET2 were quantifiable at serial dilutions ranging from SD4.0 to SD8.5
Theoretical copy number concentration was calculated from the mass concentration of the reference material (RM), and SD1.0 through SD9.5 were converted based on their dilution ratios
Summary
Horseracing began in Britain in the early 18th century and currently occurs worldwide. Recent developments in veterinary medicine have resulted in genetic doping (i.e., the illegal use of gene therapy) becoming a concern in the horseracing industry [4]. The detection of phosphothioated oligonucleotides is accomplished using mass spectrometry (MS) [6], and polymers of nucleic acids and nucleic acid analogues in this definition refer to transgenes [7,8]. In conventional doping tests, such as those targeting a low-molecular-weight compound, MS is used [11], whereas for gene-doping, MS would not be optimal for transgene detection, because the vector sequences are several kilobases long. PCR allows amplification of specific DNA segments and is used for genetic trait diagnosis and parentage testing [13,14], making it efficacious for the development of detection methods targeting gene-doping. We developed and validated a method to detect gene-doping substances using the horse EPO gene cloned in a plasmid and used as a doping substance
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