FREE SHIPPING over $40
OVER 10000 FIVE-STAR REVIEWS
5% OFF ALL SUBSCRIPTIONS
100% HAPPINESS GUARANTEE
Analyze Diet
0
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.
Get Full Text
Publication Date: 2019-03-21 PubMed ID: 30901981PubMed Central: PMC6471249DOI: 10.3390/genes10030243Google Scholar: Lookup
The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
LinkedInCopy
  • 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.

References

This article includes 17 references
  1. Wong JK, Wan TS. Doping control analyses in horseracing: a clinician's guide.. Vet J 2014 Apr;200(1):8-16.
    doi: 10.1016/j.tvjl.2014.01.006pubmed: 24485918google scholar: lookup
  2. Eenoo PV, Delbeke FT. Detection of inhaled salbutamol in equine urine by ELISA and GC/MS2.. Biomed Chromatogr 2002 Dec;16(8):513-6.
    doi: 10.1002/bmc.194pubmed: 12474214google scholar: lookup
  3. Wong CH, Leung DK, Tang FP, Wong JK, Yu NH, Wan TS. Rapid screening of anabolic steroids in horse urine with ultra-high-performance liquid chromatography/tandem mass spectrometry after chemical derivatisation.. J Chromatogr A 2012 Apr 6;1232:257-65.
    doi: 10.1016/j.chroma.2011.12.095pubmed: 22265177google scholar: lookup
  4. Wilkin T, Baoutina A, Hamilton N. Equine performance genes and the future of doping in horseracing.. Drug Test Anal 2017 Sep;9(9):1456-1471.
    doi: 10.1002/dta.2198pubmed: 28349656google scholar: lookup
  5. Goodchild J. Therapeutic oligonucleotides.. Methods Mol Biol 2011;764:1-15.
    pubmed: 21748630doi: 10.1007/978-1-61779-188-8_1google scholar: lookup
  6. Tozaki T, Karasawa K, Minamijima Y, Ishii H, Kikuchi M, Kakoi H, Hirota KI, Kusano K, Nagata SI. Detection of phosphorothioated (PS) oligonucleotides in horse plasma using a product ion (m/z 94.9362) derived from the PS moiety for doping control.. BMC Res Notes 2018 Oct 29;11(1):770.
    doi: 10.1186/s13104-018-3885-5pmc: PMC6206624pubmed: 30373660google scholar: lookup
  7. Murakami T, Sunada Y. Plasmid DNA gene therapy by electroporation: principles and recent advances.. Curr Gene Ther 2011 Dec;11(6):447-56.
    doi: 10.2174/156652311798192860pubmed: 22023474google scholar: lookup
  8. Balakrishnan B, Jayandharan GR. Basic biology of adeno-associated virus (AAV) vectors used in gene therapy.. Curr Gene Ther 2014;14(2):86-100.
    doi: 10.2174/1566523214666140302193709pubmed: 24588706google scholar: lookup
  9. Ishihara A, Shields KM, Litsky AS, Mattoon JS, Weisbrode SE, Bartlett JS, Bertone AL. Osteogenic gene regulation and relative acceleration of healing by adenoviral-mediated transfer of human BMP-2 or -6 in equine osteotomy and ostectomy models.. J Orthop Res 2008 Jun;26(6):764-71.
    doi: 10.1002/jor.20585pubmed: 18241059google scholar: lookup
  10. Kovac M, Litvin YA, Aliev RO, Zakirova EY, Rutland CS, Kiyasov AP, Rizvanov AA. 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.2017.00168pmc: PMC5641304pubmed: 29067288google scholar: lookup
  11. Moulard Y, Bailly-Chouriberry L, Boyer S, Garcia P, Popot MA, Bonnaire Y. Use of benchtop exactive high resolution and high mass accuracy orbitrap mass spectrometer for screening in horse doping control.. Anal Chim Acta 2011 Aug 26;700(1-2):126-36.
    doi: 10.1016/j.aca.2011.01.006pubmed: 21742125google scholar: lookup
  12. Perez IC, Le Guiner C, Ni W, Lyles J, Moullier P, Snyder RO. PCR-based detection of gene transfer vectors: application to gene doping surveillance.. Anal Bioanal Chem 2013 Dec;405(30):9641-53.
    doi: 10.1007/s00216-013-7264-8pubmed: 23912835google scholar: lookup
  13. Kavar T, Čeh E, Dovč P. A simplified PCR-based method for detection of gray coat color allele in horse.. Mol Cell Probes 2012 Dec;26(6):256-8.
    doi: 10.1016/j.mcp.2012.02.006pubmed: 22433981google scholar: lookup
  14. Tozaki T, Kakoi H, Mashima S, Hirota K, Hasegawa T, Ishida N, Miura N, Choi-Miura NH, Tomita M. Population study and validation of paternity testing for Thoroughbred horses by 15 microsatellite loci.. J Vet Med Sci 2001 Nov;63(11):1191-7.
    doi: 10.1292/jvms.63.1191pubmed: 11767052google scholar: lookup
  15. Morley AA. Digital PCR: A brief history.. Biomol Detect Quantif 2014 Sep;1(1):1-2.
    doi: 10.1016/j.bdq.2014.06.001pmc: PMC5129430pubmed: 27920991google scholar: lookup
  16. Tozaki T, Gamo S, Takasu M, Kikuchi M, Kakoi H, Hirota KI, Kusano K, Nagata SI. Digital PCR detection of plasmid DNA administered to the skeletal muscle of a microminipig: a model case study for gene doping detection.. BMC Res Notes 2018 Oct 10;11(1):708.
    doi: 10.1186/s13104-018-3815-6pmc: PMC6180624pubmed: 30309394google scholar: lookup
  17. Mbatha LS, Singh M. Starburst Poly(amidoamine) Dendrimer Grafted Gold Nanoparticles as a Scaffold for Folic Acid-Targeted Plasmid DNA Delivery In Vitro.. J Nanosci Nanotechnol 2019 Apr 1;19(4):1959-1970.
    doi: 10.1166/jnn.2019.15798pubmed: 30486936google scholar: lookup

Citations

This article has been cited 16 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. 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.
    doi: 10.1294/jes.32.125pubmed: 35023990google scholar: lookup
  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).
    doi: 10.3390/genes12121874pubmed: 34946824google scholar: lookup
  5. Sugasawa T, Nakano T, Fujita SI, Matsumoto Y, Ishihara G, Aoki K, Yanazawa K, Ono S, Tamai S, Manevich L, Ueda H, Ishibashi N, Tamai K, Kanki Y, Yoshida Y, Watanabe K, Takemasa T, Kawakami Y, Takekoshi K. Proof of Gene Doping in a Mouse Model with a Human Erythropoietin Gene Transferred Using an Adenoviral Vector. Genes (Basel) 2021 Aug 16;12(8).
    doi: 10.3390/genes12081249pubmed: 34440425google scholar: lookup
  6. Tozaki T, Ohnuma A, Kikuchi M, Ishige T, Kakoi H, Hirota KI, Kusano K, Nagata SI. Rare and common variant discovery by whole-genome sequencing of 101 Thoroughbred racehorses. Sci Rep 2021 Aug 6;11(1):16057.
    doi: 10.1038/s41598-021-95669-1pubmed: 34362995google scholar: lookup
  7. Tozaki T, Ohnuma A, Kikuchi M, Ishige T, Kakoi H, Hirota KI, Hamilton NA, Kusano K, Nagata SI. Whole-genome resequencing using genomic DNA extracted from horsehair roots for gene-doping control in horse sports. J Equine Sci 2020;31(4):75-83.
    doi: 10.1294/jes.31.75pubmed: 33376443google scholar: lookup
  8. Lee N, Park MJ, Song W, Jeon K, Jeong S. Currently Applied Molecular Assays for Identifying ESR1 Mutations in Patients with Advanced Breast Cancer. Int J Mol Sci 2020 Nov 20;21(22).
    doi: 10.3390/ijms21228807pubmed: 33233830google scholar: lookup
  9. Sugasawa T, Aoki K, Yanazawa K, Takekoshi K. Detection of Multiple Transgene Fragments in a Mouse Model of Gene Doping Based on Plasmid Vector Using TaqMan-qPCR Assay. Genes (Basel) 2020 Jul 6;11(7).
    doi: 10.3390/genes11070750pubmed: 32640671google scholar: lookup
  10. Tozaki T, Ohnuma A, Kikuchi M, Ishige T, Kakoi H, Hirota KI, Kusano K, Nagata SI. Microfluidic Quantitative PCR Detection of 12 Transgenes from Horse Plasma for Gene Doping Control. Genes (Basel) 2020 Apr 23;11(4).
    doi: 10.3390/genes11040457pubmed: 32340130google scholar: lookup
  11. Ueda T, Tozaki T, Nozawa S, Kinoshita K, Gawahara H. Identification of metabolomic changes in horse plasma after racing by liquid chromatography-high resolution mass spectrometry as a strategy for doping testing. J Equine Sci 2019 Sep;30(3):55-61.
    doi: 10.1294/jes.30.55pubmed: 31592223google scholar: lookup
  12. Han J, Ganguly R, Yi JY, Yun H, Jung SY, Sung C, Lee CS. Osmotically Tunable Microdroplets Enable Amplification-Free CRISPR Detection of Gene Doping. Adv Sci (Weinh) 2025 Dec;12(48):e15861.
    doi: 10.1002/advs.202515861pubmed: 41084992google scholar: lookup
  13. 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
  14. Puchalska M, Witkowska-Piłaszewicz O. Gene doping in horse racing and equine sports: Current landscape and future perspectives. Equine Vet J 2025 Mar;57(2):312-324.
    doi: 10.1111/evj.14418pubmed: 39267222google scholar: lookup
  15. 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
  16. 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
Subscribe to our newsletter
Join 99,780 horse owners like you who receive our email updates on horse health and nutrition.