Genetic Analysis Department, Laboratory of Racing Chemistry, 1731-2 Tsurutamachi, Utsunomiya, Tochigi 320-0851, Japan.
Department of Veterinary Medicine, Faculty of Applied Biological Sciences, Gifu University, 1-1 Yanagido, Gifu, Gifu 501-1193, Japan.
Genes (Basel). 2019 Mar 21;10(3):243. doi: 10.3390/genes10030243.
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 () transgenes using droplet digital PCR (ddPCR). We designed two TaqMan probe/primer sets, and the transgene was cloned into a plasmid for use as a model. We extracted the spiked 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 transgene. This represents the first study demonstrating a method for detecting the transgene in horse plasma and urine, with our results demonstrating its efficacy for promoting the control of gene-doping in the horseracing industry.
无差别基因改造以提高运动能力对人类运动和赛马行业构成重大威胁，应禁止涉及基因兴奋剂的方法，例如转基因，以确保公平。因此，迫切需要开发用于检测无差别基因改造的方法。在这里，我们使用液滴数字 PCR (ddPCR) 开发了一种高度敏感的方法来检测马促红细胞生成素 () 转基因。我们设计了两套 TaqMan 探针/引物，将 转基因克隆到质粒中作为模型。我们通过磁珠从马血浆和尿液中提取添加的 转基因，然后进行 ddPCR 扩增进行绝对定量和转基因检测。结果表明回收率很高（至少分别在血浆和尿液中分别约为 60%和 40%），表明成功检测到浓度分别大于 130 和 200 拷贝/ml 的血浆和尿液中的添加转基因。此外，通过肌肉内注射 20 毫克 转基因也成功进行了检测。这是第一项证明可在马血浆和尿液中检测 转基因的方法的研究，我们的结果表明，该方法可有效促进赛马行业基因兴奋剂的控制。
