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Open AccessArticle

Droplet Digital PCR Detection of the Erythropoietin Transgene from Horse Plasma and Urine for Gene-Doping Control

1
Genetic Analysis Department, Laboratory of Racing Chemistry, 1731-2 Tsurutamachi, Utsunomiya, Tochigi 320-0851, Japan
2
Department of Veterinary Medicine, Faculty of Applied Biological Sciences, Gifu University, 1-1 Yanagido, Gifu, Gifu 501-1193, Japan
3
Racehorse Hospital Ritto Training Center, Japan Racing Association, 1028 Misono, Ritto, Shiga 520-3085, Japan
*
Author to whom correspondence should be addressed.
Genes 2019, 10(3), 243; https://doi.org/10.3390/genes10030243
Received: 20 February 2019 / Revised: 16 March 2019 / Accepted: 18 March 2019 / Published: 21 March 2019
(This article belongs to the Special Issue Equine Genetics)
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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. View Full-Text
Keywords: genome-to-phenome; exercise traits; morphologic traits; equine genetics; domestic horse; disease mechanisms genome-to-phenome; exercise traits; morphologic traits; equine genetics; domestic horse; disease mechanisms
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Tozaki, T.; Ohnuma, A.; Takasu, M.; Kikuchi, M.; Kakoi, H.; Hirota, K.-I.; Kusano, K.; Nagata, S.-I. Droplet Digital PCR Detection of the Erythropoietin Transgene from Horse Plasma and Urine for Gene-Doping Control. Genes 2019, 10, 243.

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