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Drug testing and analysis2022; 14(9); 1587-1598; doi: 10.1002/dta.3328

Optimization and implementation of four duplex quantitative polymerase chain reaction assays for gene doping control in horseracing.

Abstract: The concern about gene doping has remained high in horseracing and other equestrian competitions. Our laboratory has previously developed a duplex quantitative polymerase chain reaction (qPCR) assay capable of detecting in equine blood the human erythropoietin (hEPO) transgene and equine tubulin α 4a (TUBA4A) gene as an internal control the latter providing quality control over DNA extraction and qPCR. This study aimed to optimize the method for routine testing of regulatory samples. The use of an automated DNA extraction system has increased the sample throughput, consistency of DNA extraction, and recovery of reference materials. The use of reduced concentration of primers and hydrolysis probe for internal control minimized their competition with transgene amplification and improved the assay sensitivity. Spike-in of an exogenous internal control at low concentration for plasma analysis has also been validated. Using the new workflow, four duplex qPCR assays have been developed for the detection of transgenes, namely, hEPO, human growth hormone (hGH), insulin-like growth factor 1 (hIGF-1), and equine EPO (eEPO). The estimated limits of detection (LODs) of each transgene were 2000 copies/mL of blood and 200 copies/mL of plasma. This method could detect the presence of transgene in blood and plasma collected from a horse administered intramuscularly (IM) with recombinant adeno-associated virus (rAAV) carrying the hEPO transgene. A longer detection time was observed in blood than in plasma. The methods have been applied to the screening of over a thousand official racehorse samples since June 2020 for the presence of these transgenes.
© 2022 John Wiley & Sons Ltd.
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Publication Date: 2022-06-06 PubMed ID: 35633307DOI: 10.1002/dta.3328Google Scholar: Lookup
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  • Journal Article

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 focused on improving methods for detecting gene doping in horserace horses through the development of an enhanced real-time polymerase chain reaction (qPCR) testing mechanism. The refined technique, which was applied to over a thousand official racehorse samples, resulted in the successful detection of specific illicit transgenes in blood and plasma.

Introduction

  • The research focuses on gene doping, a prevalent concern in horseracing and other equestrian sports.
  • The authors previously created a duplex qPCR test that could identify the human erythropoietin (hEPO) transgene and equine tubulin α 4a (TUBA4A) gene in horse blood. The TUBA4A gene acted as an internal control, providing quality assurance for DNA extraction and qPCR.
  • This latest study worked to enhance this testing method for routine usage in regulatory sample checking.

Methods

  • The researchers used an automated DNA extraction system to increase the efficacy and consistency of DNA extraction, and to improve the recovery of reference materials.
  • They also reduced the concentration of primers and the hydrolysis probe for the internal control to lessen their competition with transgene amplification, which allowed for greater test sensitivity.
  • The study validated the spike-in of a low concentration exogenous internal control for plasma analysis.

Results

  • Following the new workflow, four duplex qPCR tests were designed for the detection of the following transgenes: hEPO, human growth hormone (hGH), insulin-like growth factor 1 (hIGF-1), and equine EPO (eEPO).
  • The estimated limits of detection (LODs) of each transgene were 2000 copies/mL in blood and 200 copies/mL in plasma.
  • The refined method was successfully able to identify the presence of the hEPO transgene in blood and plasma from a horse that had been intramuscularly administered with a recombinant adeno-associated virus (rAAV) carrying the hEPO transgene. It was also noted that the detection time was longer in blood than in plasma.

Conclusion

  • Since June 2020, the enhanced methods have been applied in the screening of over a thousand racehorse samples for the presence of these transgenes.

Cite This Article

APA
Cheung HW, Wong KS, Lin VYC, Farrington AF, Bond AJ, Wan TSM, Ho ENM. (2022). Optimization and implementation of four duplex quantitative polymerase chain reaction assays for gene doping control in horseracing. Drug Test Anal, 14(9), 1587-1598. https://doi.org/10.1002/dta.3328

Publication

Drug testing and analysis
ISSN: 1942-7611
NlmUniqueID: 101483449
Country: England
Language: English
Volume: 14
Issue: 9
Pages: 1587-1598

Researcher Affiliations

Cheung, Hiu Wing
  • Racing Laboratory, The Hong Kong Jockey Club, Sha Tin Racecourse, Sha Tin, Hong Kong, China.
Wong, Kin-Sing
  • Racing Laboratory, The Hong Kong Jockey Club, Sha Tin Racecourse, Sha Tin, Hong Kong, China.
Lin, Venus Y C
  • Racing Laboratory, The Hong Kong Jockey Club, Sha Tin Racecourse, Sha Tin, Hong Kong, China.
Farrington, Adrian F
  • Veterinary Clinical Services, The Hong Kong Jockey Club, Sha Tin Racecourse, Sha Tin, Hong Kong, China.
Bond, Amanda J
  • Equestrian Affairs, The Hong Kong Jockey Club, Sha Tin Racecourse, Sha Tin, Hong Kong, China.
Wan, Terence S M
  • Racing Laboratory, The Hong Kong Jockey Club, Sha Tin Racecourse, Sha Tin, Hong Kong, China.
Ho, Emmie N M
  • Racing Laboratory, The Hong Kong Jockey Club, Sha Tin Racecourse, Sha Tin, Hong Kong, China.

MeSH Terms

  • Animals
  • DNA
  • DNA Primers
  • Dependovirus / genetics
  • Doping in Sports / prevention & control
  • Horses / genetics
  • Humans
  • Polymerase Chain Reaction / methods
  • Recombinant Proteins
  • Transgenes

References

This article includes 29 references
  1. Papanikolaou E, Bosio A. The promise and the hope of gene therapy.. Front Genome Ed 2021;3:618346.
    doi: 10.3389/fgeed.2021.618346google scholar: lookup
  2. Shahryari A, Saghaeian Jazi M, Mohammadi S. Development and clinical translation of approved gene therapy products for genetic disorders.. Front Genet 2019;10:868.
    doi: 10.3389/fgene.2019.00868google scholar: lookup
  3. Moss KL, Jiang Z, Dodson ME. Sustained Interleukin-10 transgene expression following intra-articular AAV5-IL-10 administration to horses.. Hum Gene Ther 2020;31(1-2):110-118.
    doi: 10.1089/hum.2019.195google scholar: lookup
  4. Li C, Samulski RJ. Engineering adeno-associated virus vectors for gene therapy.. Nat Rev Genet 2020;21(4):255-272.
    doi: 10.1038/s41576-019-0205-4google scholar: lookup
  5. Tozaki T, Hamilton NA. Control of gene doping in human and horse sports.. Gene Ther 2021;29(3-4):107-112.
    doi: 10.1038/s41434-021-00267-5google scholar: lookup
  6. Wilkin T, Baoutina A, Hamilton N. Equine performance genes and the future of doping in horseracing.. Drug Test Anal 2017;9(9):1456-1471.
    doi: 10.1002/dta.2198google scholar: lookup
  7. Baoutina A, Alexander IE, Rasko JE, Emslie KR. Potential use of gene transfer in athletic performance enhancement.. Mol Ther 2007;15(10):1751-1766.
    doi: 10.1038/sj.mt.6300278google scholar: lookup
  8. International Federation of Horseracing Authorities. International agreement on breeding, racing and wagering and appendixes. Article 6B. .
  9. Fédération Équestre Internationale. Equine 2021 Veterinary Regulations.. .
  10. Kovac M, Litvin YA, Aliev RO. Gene therapy using plasmid DNA encoding vascular endothelial growth factor 164 and fibroblast growth factor 2 genes for the treatment of horse tendinitis and desmitis: case reports.. Front Vet Sci 2017;4:168.
    doi: 10.3389/fvets.2017.00168google scholar: lookup
  11. Maniego J, Pesko B, Habershon-Butcher J. Screening for gene doping transgenes in horses via the use of massively parallel sequencing.. Gene Ther 2022;29(5):236-246.
    doi: 10.1038/s41434-021-00279-1google scholar: lookup
  12. Mason JB, Gurda BL, van Wettere A, Engiles JB, Wilson JM, Richardson DW. Delivery and evaluation of recombinant adeno-associated viral vectors in the equine distal extremity for the treatment of laminitis.. Equine Vet J 2017;49(1):79-86.
    doi: 10.1111/evj.12547google scholar: lookup
  13. Tozaki T, Ohnuma A, Takasu M. Droplet digital PCR detection of the erythropoietin transgene from horse plasma and urine for gene-doping control.. Genes (Basel) 2019;10(3):243.
    doi: 10.3390/genes10030243google scholar: lookup
  14. Haughan J, Jiang Z, Stefanovski D, Moss KL, Ortved KF, Robinson MA. Detection of intra-articular gene therapy in horses using quantitative real time PCR in synovial fluid and plasma.. Drug Test Anal 2020;12(6):743-751.
    doi: 10.1002/dta.2785google scholar: lookup
  15. Jiang Z, Haughan J, Moss KL, Stefanovski D, Ortved KF, Robinson MA. A quantitative PCR screening method for adeno-associated viral vector 2-mediated gene doping.. Drug Test Anal 2021;14(5):963-972.
    doi: 10.1002/dta.3152google scholar: lookup
  16. Tozaki T, Ohnuma A, Kikuchi M. Microfluidic quantitative PCR detection of 12 transgenes from horse plasma for gene doping control.. Genes 2020;11(4):457.
    doi: 10.3390/genes11040457google scholar: lookup
  17. Tozaki T, Ohnuma A, Takasu M. Detection of non-targeted transgenes by whole-genome resequencing for gene-doping control.. Gene Ther 2021;28(3-4):199-205.
    doi: 10.1038/s41434-020-00185-ygoogle scholar: lookup
  18. Cheung HW, Wong KS, Lin VYC, Wan TSM, Ho ENM. A duplex qPCR assay for human erythropoietin (EPO) transgene to control gene doping in horses.. Drug Test Anal 2021;13(1):113-121.
    doi: 10.1002/dta.2907google scholar: lookup
  19. Bray MS, Hagberg JM, Perusse L. The human gene map for performance and health-related fitness phenotypes: the 2006-2007 update.. Med Sci Sports Exerc 2009;41(1):35-73.
    doi: 10.1249/mss.0b013e3181844179google scholar: lookup
  20. Holt RIG, Ho KKY. The use and abuse of growth hormone in sports.. Endocr Rev 2019;40(4):1163-1185.
    doi: 10.1210/er.2018-00265google scholar: lookup
  21. International Organization for Standardization. ISO 20395:2019 Biotechnology-requirements for evaluating the performance of quantification methods for nucleic acid target sequences-qPCR and dPCR.. 2019.
  22. Enna SJ, Bylund DB. xPharm: The Comprehensive Pharmacology Reference.. 2008.
  23. Favre D, Blouin V, Provost N. Lack of an immune response against the tetracycline-dependent transactivator correlates with long-term doxycycline-regulated transgene expression in nonhuman primates after intramuscular injection of recombinant adeno-associated virus.. J Virol 2002;76(22):11605-11611.
    doi: 10.1128/jvi.76.22.11605-11611.2002google scholar: lookup
  24. Yu NH, Ho EN, Wan TS, Wong AS. Doping control analysis of recombinant human erythropoietin, darbepoetin alfa and methoxy polyethylene glycol-epoetin beta in equine plasma by nano-liquid chromatography-tandem mass spectrometry.. Anal Bioanal Chem 2010;396(7):2513-2521.
    doi: 10.1007/s00216-010-3455-8google scholar: lookup
  25. International Laboratory Accreditation Cooperation. ILAC G7:04/2021 Accreditation Requirements and Operating Criteria for Horseracing Laboratories.. 2021.
  26. Ni W, le Guiner C, Gernoux G, Penaud-Budloo M, Moullier P, Snyder RO. Longevity of rAAV vector and plasmid DNA in blood after intramuscular injection in nonhuman primates: implications for gene doping.. Gene Ther 2011;18(7):709-718.
    doi: 10.1038/gt.2011.19google scholar: lookup
  27. Favre D, Provost N, Blouin V. Immediate and long-term safety of recombinant adeno-associated virus injection into the nonhuman primate muscle.. Mol Ther 2001;4(6):559-566.
    doi: 10.1006/mthe.2001.0494google scholar: lookup
  28. Chenuaud P, Larcher T, Rabinowitz JE. Autoimmune anemia in macaques following erythropoietin gene therapy.. Blood 2004;103(9):3303-3304.
    doi: 10.1182/blood-2003-11-3845google scholar: lookup
  29. Gao G, Lebherz C, Weiner DJ. Erythropoietin gene therapy leads to autoimmune anemia in macaques.. Blood 2004;103(9):3300-3302.
    doi: 10.1182/blood-2003-11-3852google scholar: lookup

Citations

This article has been cited 6 times.
  1. Tozaki T, Ohnuma A, Kikuchi M, Ishige T, Kakoi H, Hirota KI, Takahashi Y, Nagata SI. Investigation of optimal procedures for storage and use of plasma samples suitable for gene doping tests. J Equine Sci 2023 Jun;34(2):21-27.
    doi: 10.1294/jes.34.21pubmed: 37405066google scholar: lookup
  2. Tozaki T, Ohnuma A, Nakamura K, Hano K, Takasu M, Takahashi Y, Tamura N, Sato F, Shimizu K, Kikuchi M, Ishige T, Kakoi H, Hirota KI, Hamilton NA, Nagata SI. Detection of Indiscriminate Genetic Manipulation in Thoroughbred Racehorses by Targeted Resequencing for Gene-Doping Control. Genes (Basel) 2022 Sep 4;13(9).
    doi: 10.3390/genes13091589pubmed: 36140757google scholar: lookup
  3. Naumann N, Do C, Vollmert C, Krajina M, Thomas A, Cheung HW, Wong KS, Wan TSM, Ho ENM, Thevis M. Multiplex detection of seven transgenes for human gene doping analysis. Sci Rep 2025 Jun 20;15(1):20219.
    doi: 10.1038/s41598-025-06677-4pubmed: 40542126google scholar: lookup
  4. Maniego J, Harding C, Habershon-Butcher J, Hincks P, Stewart G, Proudman C, Ryder E. Detection of transgenes in equine dried blood spots using digital PCR and qPCR for gene doping control. Drug Test Anal 2025 May;17(5):626-633.
    doi: 10.1002/dta.3755pubmed: 38992991google scholar: lookup
  5. Maniego J, Harding C, Habershon-Butcher J, Hincks P, Ryder E. Administration and detection of a multi-target rAAV gene doping vector in horses using multiple matrices and molecular techniques. Gene Ther 2024 Sep;31(9-10):477-488.
    doi: 10.1038/s41434-024-00462-0pubmed: 38972888google scholar: lookup
  6. Wilkin T, Hamilton NA, Cawley AT, Bhat S, Baoutina A. PCR-Based Equine Gene Doping Test for the Australian Horseracing Industry. Int J Mol Sci 2024 Feb 22;25(5).
    doi: 10.3390/ijms25052570pubmed: 38473816google scholar: lookup
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