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Drug testing and analysis2024; doi: 10.1002/dta.3745

A method for detecting gene doping in horse sports without DNA extraction.

Abstract: Gene doping is prohibited in horse sports and can involve the administration of exogenous genes, called transgenes, to postnatal animals. Quantitative polymerase chain reaction (qPCR) methods have been developed to detect gene doping; however, these generally require DNA extraction from the plasma prior to qPCR. In this study, we developed two methods, direct droplet digital PCR (ddPCR) and nested ddPCR, to detect the equine erythropoietin (EPO) transgene without DNA extraction. Direct ddPCR used pretreated plasma and PCR to detect the EPO transgene spiked at 10 copies/μL. Nested ddPCR utilised pre-amplification using nontreated plasma, purification of PCR products and PCR to detect the EPO transgene spiked at 1 copy/μL in plasma. These methods successfully detected the EPO transgene after intramuscular injection into horses. Since each method has different detection sensitivity, the combined use of direct ddPCR for screening and nested ddPCR for confirmation may complement each other and prevent the occurrence of false positives, allowing the reliable detection of gene-doped substances. One advantage of these methods is the small amount of sample required, approximately 2.2-5.0 μl, owing to the lack of a DNA extraction step. Therefore, these tests could be applied to small volume samples as an alternative to conventional gene doping tests.
© 2024 John Wiley & Sons Ltd.
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Publication Date: 2024-06-09 PubMed ID: 38853330DOI: 10.1002/dta.3745Google Scholar: Lookup
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Summary

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The research article discusses a new method developed to detect illegal gene manipulation, or doping, in horse sports without the need for DNA extraction. By using a Direct Droplet Digital PCR (ddPCR) and nested ddPCR, scientists were able to detect equine erythropoietin (EPO) transgenes from small sample quantities.

Background and Objective

  • Gene doping – introducing exogenous genes or changing existing ones to enhance an animal’s competitive abilities – is strongly prohibited in sports. These transgenes are difficult to detect in a traditional drug screening.
  • Existing methods to detect gene doping rely on DNA extraction from the blood plasma, which is time-consuming and needs larger sample volumes. The study aimed to develop a new method that could bypass DNA extraction and allow the detection of transgenes with increased efficiency.

    • Method

    • The researchers designed two methods to detect the EPO transgenes, namely direct droplet digital PCR (ddPCR) and nested ddPCR.
    • The direct ddPCR is used to detect the EPO transgene present at a concentration of 10 copies per microliter using pretreated plasma and PCR.
    • The nested ddPCR, on the other hand, was used to detect the EPO transgene at even lower concentrations, i.e. 1 copy per microliter. This method involved pre-amplification using non-treated plasma, purification of PCR products, and then PCR.

      • Results and Conclusion

      • The study was successful in detecting the EPO transgene in horses after being administered an intramuscular injection, by using the mentioned methods.
      • Each method has a different detection sensitivity which, when used in combination, can prevent false positives and offer a reliable detection mechanism.
      • A significant advantage of these methods is the small sample volume required, between 2.2 – 5.0 μL, since there is no need for DNA extraction. This makes them a highly feasible alternative to conventional gene doping test.

Cite This Article

APA
Furukawa R, Tozaki T, Kikuchi M, Ishige T, Takahashi Y, Fukui E, Kakoi H. (2024). A method for detecting gene doping in horse sports without DNA extraction. Drug Test Anal. https://doi.org/10.1002/dta.3745

Publication

Drug testing and analysis
ISSN: 1942-7611
NlmUniqueID: 101483449
Country: England
Language: English

Researcher Affiliations

Furukawa, Risako
  • Genetic Analysis Department, Laboratory of Racing Chemistry, Utsunomiya, Tochigi, Japan.
Tozaki, Teruaki
  • Genetic Analysis Department, Laboratory of Racing Chemistry, Utsunomiya, Tochigi, Japan.
Kikuchi, Mio
  • Genetic Analysis Department, Laboratory of Racing Chemistry, Utsunomiya, Tochigi, Japan.
Ishige, Taichiro
  • Genetic Analysis Department, Laboratory of Racing Chemistry, Utsunomiya, Tochigi, Japan.
Takahashi, Yuji
  • Equine Research Institute, Japan Racing Association, Shimotsuke, Tochigi, Japan.
Fukui, Emiko
  • School of Agriculture, Utsunomiya University, Utsunomiya, Tochigi, Japan.
Kakoi, Hironaga
  • Genetic Analysis Department, Laboratory of Racing Chemistry, Utsunomiya, Tochigi, Japan.

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Citations

This article has been cited 2 times.
  1. Maehara K, Hirokawa A, Watanabe H, Otani N, Wan J, Shirai T, Takemasa T, Watanabe K, Nishi T, Sato K, Shimmura S, Nguyen KDM, Takahashi Y, Sugasawa T. Development of Detection Method Using Dried Blood Spot with Next-Generation Sequencing and LabDroid for Gene Doping Control. Int J Mol Sci 2025 Jun 26;26(13).
    doi: 10.3390/ijms26136129pubmed: 40649907google scholar: lookup
  2. Thomas A, Walpurgis K, Naumann N, Piper T, Thevis M. Bioanalytical methods in doping controls: a review. Bioanalysis 2025 Mar;17(5):359-370.
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