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Drug testing and analysis2026; doi: 10.1002/dta.70103

Towards a Comprehensive Screening Approach for Detecting Gene Doping in Racehorses and Sport Horses by Targeting Common and Intrinsic Plasmid Sequences.

Abstract: The evolution of biotechnology and gene therapy has unfortunately facilitated the emergence of gene doping. Those misused applications are prohibited in equestrian sports (International Equestrian Federation (FEI)) and in horseracing (International Federation of Horseracing Authorities [IFHA]), reflecting major concerns regarding horse welfare and the integrity of competitions. Among the biotechnological tools that could be misused, eukaryotic expression plasmids represent a particular threat due to their ease of production, customisable design and ability to carry a wide range of performance transgenes. Current detection methods are mainly based on the identification of these transgenes, but if the sequence is unknown or modified, administration may not be detected. The aim of this work was to overcome this limitation by developing an untargeted transgene screening approach in equine plasma. A database of 349 mammalian expression plasmids was compiled, allowing identification of four conserved targets: the ampicillin resistance gene (AmpR), the cytomegalovirus promoter (pCMV) and two unannotated sequences between the features, which together covered 100% of the plasmids database. A set of PCR hydrolysis probe assays targeting these sequences has been developed and exhibited excellent efficiency, linearity and robustness. The method was able to discriminate, in plasma, 100% of suspicious samples from background noise down to 1200 copies/mL. Following plasmid administration to a horse, all four targets remained detectable up to 48 h using qPCR and dPCR, demonstrating the proof of concept. Overall, this work presented a sensitive, reliable and transgene-independent method that strengthens plasmid detection and reinforces gene doping control in equine athletes.
© 2026 John Wiley & Sons Ltd.
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Publication Date: 2026-06-12 PubMed ID: 42285585DOI: 10.1002/dta.70103Google Scholar: Lookup
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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.

Overview

  • This study developed a novel, comprehensive method to detect gene doping in racehorses and sport horses by targeting common plasmid DNA sequences rather than relying on specific transgene detection, thereby improving the ability to identify illegal gene doping regardless of the transgene used.

Background and Motivation

  • Gene doping refers to the misuse of gene therapy techniques to enhance athletic performance, which is prohibited in equestrian sports and horseracing due to concerns about horse welfare and fair competition.
  • Recent advances in biotechnology and gene therapy have made gene doping easier, particularly through the use of eukaryotic expression plasmids—a type of DNA molecule used to carry and express performance-enhancing genes.
  • These plasmids are attractive for gene doping because they are easy to produce, customizable in design, and can carry a wide variety of transgenes.
  • Conventional detection methods focus on identifying the specific transgene sequences administered; however, these methods can fail if gene sequences are unknown or deliberately modified to evade detection.

Research Aim

  • To develop a transgene-independent, broad screening method capable of detecting eukaryotic expression plasmids used in gene doping, regardless of the specific transgene sequence.
  • Specifically, to identify conserved plasmid regions common across many plasmids and create assays to detect these regions in equine plasma samples.

Methodology

  • Compiled a database consisting of 349 mammalian expression plasmids to analyze and identify commonly shared sequences across these plasmids.
  • Identified four conserved plasmid targets that appeared in 100% of the plasmids in the database:
    • Ampicillin resistance gene (AmpR)
    • Cytomegalovirus promoter (pCMV)
    • Two unannotated sequences located between known features
  • Developed PCR (polymerase chain reaction) hydrolysis probe assays targeting these four sequences to detect the presence of plasmid DNA.
  • Validated the PCR assays through tests verifying:
    • Efficiency (ability to amplify target DNA)
    • Linearity (consistent detection over a range of DNA concentrations)
    • Robustness (reliability under different test conditions)
  • Conducted sensitivity testing in plasma samples to differentiate true plasmid signals from background noise, achieving detection down to 1200 copies per milliliter of plasma.
  • Performed in vivo proof-of-concept by administering plasmids to a horse and monitoring detection via quantitative PCR (qPCR) and digital PCR (dPCR), which confirmed plasmid presence up to 48 hours post-administration.

Key Findings

  • All 349 plasmids in the database contained at least one of the four target sequences, indicating that the assay covers the full range of common expression plasmids used in mammalian gene therapy.
  • The developed PCR assays were sensitive enough to detect plasmid DNA at low levels within plasma samples, providing a clear distinction between true positives and background noise.
  • Detection post-plasmid administration demonstrated the practical applicability of this method in live animals, confirming it can monitor gene doping events for a significant window of time.

Implications

  • This transgene-independent screening method enhances the capacity to detect gene doping regardless of the specific gene sequence used, addressing the major limitation of existing detection approaches.
  • The method strengthens the oversight and enforcement mechanisms for gene doping in horse racing and equestrian sports, helping to safeguard both animal welfare and competition integrity.
  • The approach could potentially be adapted for gene doping detection in other animals or even human sports industries where gene doping is a concern.

Conclusion

  • The study successfully demonstrated a novel and comprehensive gene doping detection strategy targeting conserved plasmid sequences rather than specific transgenes.
  • This method offers a sensitive, robust, and reliable tool to improve anti-doping controls within equine sports, contributing significantly to the fight against illicit gene modification practices.

Cite This Article

APA
Paz A, Dhorne-Pollet S, Loup B, André F, Garcia P, Barrey E, Bailly-Chouriberry L. (2026). Towards a Comprehensive Screening Approach for Detecting Gene Doping in Racehorses and Sport Horses by Targeting Common and Intrinsic Plasmid Sequences. Drug Test Anal. https://doi.org/10.1002/dta.70103

Publication

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

Researcher Affiliations

Paz, Angèle
  • GIE LCH, Laboratoire des Courses Hippiques, Verrières-le-Buisson, France.
  • Université Paris-Saclay, INRAE, AgroParisTech, GABI UMR1313, Jouy-en-Josas, France.
Dhorne-Pollet, Sophie
  • Université Paris-Saclay, INRAE, AgroParisTech, GABI UMR1313, Jouy-en-Josas, France.
Loup, Benoit
  • GIE LCH, Laboratoire des Courses Hippiques, Verrières-le-Buisson, France.
André, François
  • GIE LCH, Laboratoire des Courses Hippiques, Verrières-le-Buisson, France.
Garcia, Patrice
  • GIE LCH, Laboratoire des Courses Hippiques, Verrières-le-Buisson, France.
Barrey, Eric
  • Université Paris-Saclay, INRAE, AgroParisTech, GABI UMR1313, Jouy-en-Josas, France.
Bailly-Chouriberry, Ludovic
  • GIE LCH, Laboratoire des Courses Hippiques, Verrières-le-Buisson, France.

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