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Home / Equine Research Bank / Genes (Basel) / Droplet Digital PCR Detection of the Erythropoietin Transgen...
Genes2019; 10(3); 243; doi: 10.3390/genes10030243

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

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 () 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.
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Publication Date: 2019-03-21 PubMed ID: 30901981PubMed Central: PMC6471249DOI: 10.3390/genes10030243Google Scholar: Lookup
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  • Journal Article
  • Research Support
  • Non-U.S. Gov't

Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

This research paper focuses on developing a method to detect gene-doping in horses through the detection of erythropoietin (EPO) transgenes in horse urine and plasma using a technique called droplet digital PCR. The results indicate it could be effectively used to regulate fairness in the horseracing industry.

Objective of the Research

  • The researchers aim to develop a sensitive and effective method to detect gene-doping in horseracing, a growing concern in the industry due to its potential use in illicitly enhancing a horse’s athletic abilities.
  • The method involves the detection of horse erythropoietin (EPO) transgenes from horse plasma and urine using droplet digital PCR (ddPCR). EPO is a hormone that promotes the production of red blood cells and its presence could suggest genetic manipulation aimed at enhancing performance.

Methodology

  • The team designed two TaqMan probe/primer sets, essential tools for amplifying the target DNA sequence.
  • The EPO transgene was cloned into a plasmid, a DNA molecule that’s often used in laboratories as a vehicle for amplifying and making multiple copies of the target DNA.
  • This plasmid with the EPO transgene was then mixed (‘spiked’) with horse plasma and urine samples, which were prepared using magnetic beads—a common laboratory technique for isolating particular biomolecules from a sample.
  • After sample preparation, ddPCR was used to amplify and quantify the EPO transgene for detection.

Findings

  • The researchers found high recovery rates of the transgene from both the plasma and urine samples, indicating successful detection of the spiked EPO transgene. The recovery rate was about 60% in plasma and 40% in urine.
  • The EPO transgene was detectable at concentrations greater than 130 and 200 copies per milliliter of plasma and urine, respectively.
  • The detection method was also tested in a more real-life scenario by injecting a horse with the EPO transgene. The researchers were able to successfully detect the EPO transgene in this setup as well.

Implications

  • This research represents the first study that demonstrates a method for detecting an EPO transgene in horse plasma and urine, which could provide a powerful tool in the fight against gene doping in horse racing.
  • The demonstrated efficacy of this method could lead to its potential use in promoting the control of gene-doping in the horseracing industry, thus ensuring a fair and level playing field for all participants.

Cite This Article

APA
Tozaki T, Ohnuma A, Takasu M, Kikuchi M, Kakoi H, Hirota KI, Kusano K, Nagata SI. (2019). Droplet Digital PCR Detection of the Erythropoietin Transgene from Horse Plasma and Urine for Gene-Doping Control. Genes (Basel), 10(3), 243. https://doi.org/10.3390/genes10030243

Publication

Genes
ISSN: 2073-4425
NlmUniqueID: 101551097
Country: Switzerland
Language: English
Volume: 10
Issue: 3
PII: 243

Researcher Affiliations

Tozaki, Teruaki
  • Genetic Analysis Department, Laboratory of Racing Chemistry, 1731-2 Tsurutamachi, Utsunomiya, Tochigi 320-0851, Japan. ttozaki@lrc.or.jp.
  • Department of Veterinary Medicine, Faculty of Applied Biological Sciences, Gifu University, 1-1 Yanagido, Gifu, Gifu 501-1193, Japan. ttozaki@lrc.or.jp.
Ohnuma, Aoi
  • Genetic Analysis Department, Laboratory of Racing Chemistry, 1731-2 Tsurutamachi, Utsunomiya, Tochigi 320-0851, Japan. a-ohnuma@lrc.or.jp.
Takasu, Masaki
  • Department of Veterinary Medicine, Faculty of Applied Biological Sciences, Gifu University, 1-1 Yanagido, Gifu, Gifu 501-1193, Japan. m-kikuchi@lrc.or.jp.
Kikuchi, Mio
  • Genetic Analysis Department, Laboratory of Racing Chemistry, 1731-2 Tsurutamachi, Utsunomiya, Tochigi 320-0851, Japan. h-kakoi@lrc.or.jp.
Kakoi, Hironaga
  • Genetic Analysis Department, Laboratory of Racing Chemistry, 1731-2 Tsurutamachi, Utsunomiya, Tochigi 320-0851, Japan. k-hirota@lrc.or.jp.
Hirota, Kei-Ichi
  • Genetic Analysis Department, Laboratory of Racing Chemistry, 1731-2 Tsurutamachi, Utsunomiya, Tochigi 320-0851, Japan. s-nagata@lrc.or.jp.
Kusano, Kanichi
  • Racehorse Hospital Ritto Training Center, Japan Racing Association, 1028 Misono, Ritto, Shiga 520-3085, Japan. takasu@gifu-u.ac.jp.
Nagata, Shun-Ichi
  • Genetic Analysis Department, Laboratory of Racing Chemistry, 1731-2 Tsurutamachi, Utsunomiya, Tochigi 320-0851, Japan. Kanichi_Kusano@jra.go.jp.

MeSH Terms

  • Animals
  • Doping in Sports
  • Erythropoietin / blood
  • Erythropoietin / genetics
  • Erythropoietin / urine
  • Horses / blood
  • Horses / genetics
  • Horses / urine
  • Humans
  • Plasmids / genetics
  • Polymerase Chain Reaction / methods
  • Transgenes

Conflict of Interest Statement

There are no competing interests including patents, products in development, or marketed products to declare in relationship to this work.

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Citations

This article has been cited 11 times.
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  3. Tozaki T, Ohnuma A, Kikuchi M, Ishige T, Kakoi H, Hirota KI, Kusano K, Nagata SI. Design and storage stability of reference materials for microfluidic quantitative PCR-based equine gene doping tests.. J Equine Sci 2021 Dec;32(4):125-134.
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  4. Dahlgren AR, Knych HK, Arthur RM, Durbin-Johnson BP, Finno CJ. Transcriptomic Markers of Recombinant Human Erythropoietin Micro-Dosing in Thoroughbred Horses.. Genes (Basel) 2021 Nov 24;12(12).
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