Abstract
Gene doping, an activity which abuses and misuses gene therapy, is a major concern in sports and horseracing industries. Effective methods capable of detecting and monitoring gene doping are urgently needed. Although several PCR-based methods that detect transgenes have been developed, many of them focus only on a single transgene. However, numerous genes associated with athletic ability may be potential gene-doping material. Here, we developed a detection method that targets multiple transgenes. We targeted 12 genes that may be associated with athletic performance and designed two TaqMan probe/primer sets for each one. A panel of 24 assays was prepared and detected via a microfluidic quantitative PCR (MFQPCR) system using integrated fluidic circuits (IFCs). The limit of detection of the panel was 6.25 copy/μL. Amplification-specificity was validated using several concentrations of reference materials and animal genomic DNA, leading to specific detection. In addition, target-specific detection was successfully achieved in a horse administered 20 mg of the EPO transgene via MFQPCR. Therefore, MFQPCR may be considered a suitable method for multiple-target detection in gene-doping control. To our knowledge, this is the first application of microfluidic qPCR (MFQPCR) for gene-doping control in horseracing.
Highlights
Horses are being increasingly used in sports, with particular reference to activities such as horseracing, riding and polo
To the best of our knowledge, the method described here represents the first application of microfluidic quantitative PCR (qPCR) (MFQPCR) for the purpose of controlling gene doping in horseracing
Over 20,000 genes are annotated in the horse genome, allowing many genes to be targeted as transgenes for the express purpose of gene doping [33]
Summary
Horses are being increasingly used in sports, with particular reference to activities such as horseracing, riding and polo. Careful management at various levels is required to ensure fair practice in sports. Horseracing has led to the selection of stallions and blood mares that can be used to produce the generation of racehorses. Doping control is one important aspect [1]. Racing authorities and event organizers are responsible for doping control in horseracing, as well as in other equestrian sports. Prohibited substances mainly include low molecular weight compounds, such as β-agonists and steroids. These substances are detected via mass spectrometry and enzyme-linked immunosorbent assay (ELISA) [2,3]. The development of effective detection methods is expected to deter doping
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