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.
Publication Date: 2024-06-09 PubMed ID: 38853330DOI: 10.1002/dta.3745Google 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.
- 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.
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.
- 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.
- 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.
Method
Results and Conclusion
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
Researcher Affiliations
- Genetic Analysis Department, Laboratory of Racing Chemistry, Utsunomiya, Tochigi, Japan.
- Genetic Analysis Department, Laboratory of Racing Chemistry, Utsunomiya, Tochigi, Japan.
- Genetic Analysis Department, Laboratory of Racing Chemistry, Utsunomiya, Tochigi, Japan.
- Genetic Analysis Department, Laboratory of Racing Chemistry, Utsunomiya, Tochigi, Japan.
- Equine Research Institute, Japan Racing Association, Shimotsuke, Tochigi, Japan.
- School of Agriculture, Utsunomiya University, Utsunomiya, Tochigi, Japan.
- Genetic Analysis Department, Laboratory of Racing Chemistry, Utsunomiya, Tochigi, Japan.
References
This article includes 30 references
- Tozaki T, Hamilton NA. Control of gene doping in human and horse sports. Gene Ther 2022;29(3–4):107‐112.
- Wilkin T, Baoutina A, Hamilton N. Equine performance genes and the future of doping in horseracing. Drug Test Anal 2017;9(9):1456‐1471.
- 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.
- Budsuren U, Ulaangerel T, Shen Y. MSTN regulatory network in Mongolian horse muscle satellite cells revealed with miRNA interference technologies. Genes (Basel) 2022;13(10):1836.
- Moro LN, Viale DL, Bastón JI. Generation of myostatin edited horse embryos using CRISPR/Cas9 technology and somatic cell nuclear transfer. Sci Rep 2020;10(1):15587.
- Mançanares ACF, Cabezas J, Manríquez J. Edition of prostaglandin E2 receptors EP2 and EP4 by CRISPR/Cas9 technology in equine adipose mesenchymal stem cells. Animals (Basel) 2020;10(6):1078.
- The International Federation of Horseracing Authorities. Article 6, the international agreement on breeding, racing and wagering, 2023 edition. https://www.ifhaonline.org/resources/ifAgreement.pdf. Accessed March 19, 2024.
- Haughan J, Ortved KF, Robinson MA. Administration and detection of gene therapy in horses: a systematic review. Drug Test Anal 2023;15(2):143‐162.
- Neuhaus CP, Parent B. Gene doping‐in animals? Ethical issues at the intersection of animal use, gene editing, and sports ethics. Camb Q Healthc Ethics 2019;28(1):26‐39.
- The International Federation of Horseracing Authorities. Article 12, the international agreement on breeding, racing and wagering, 2023 edition. https://www.ifhaonline.org/resources/ifAgreement.pdf. Accessed March 19, 2024.
- Birzniece V. Doping in sport: effects, harm and misconceptions. Intern Med J 2015;45(3):239‐248.
- Aimaletdinov A, Mindubaeva G, Khalikova S. Application of gene therapy in the treatment of superficial digital flexor tendon injury in horses. Open Vet J 2020;10(3):261‐266.
- Kovac M, Litvin YA, Aliev RO. Gene therapy using plasmid DNA encoding VEGF164 and FGF2 genes: a novel treatment of naturally occurring tendinitis and desmitis in horses. Front Pharmacol 2018;9:978.
- 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.
- Cheung HW, Wong KS, Lin VYC. Optimization and implementation of four duplex quantitative polymerase chain reaction assays for gene doping control in horseracing. Drug Test Anal 2022;14(9):1587‐1598.
- 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 2022;14(5):963‐972.
- 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.
- Tozaki T, Ohnuma A, Kikuchi M. Microfluidic quantitative PCR detection of 12 transgenes from horse plasma for gene doping control. Genes (Basel) 2020;11(4):457.
- Tozaki T, Ohnuma A, Hamilton NA. Low‐copy transgene detection using nested digital polymerase chain reaction for gene‐doping control. Drug Test Anal 2022;14(2):382‐387.
- 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;25(5):2570.
- Wong KS, Cheung HW, Szeto CWL, Tsang CYN, Wan TSM, Ho ENM. A multiplex qPCR assay for transgenes detection: a novel approach for gene doping control in horseracing using conventional laboratory setup. Drug Test Anal 2023;15(8):879‐888.
- Ohnuma A, Tozaki T, Kikuchi M. Multiplex detection of transgenes using πCode technology for gene doping control. Anal Chem 2023;95(27):10149‐10154.
- 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.
- Maniego J, Pesko B, Hincks P. Direct sequence confirmation of qPCR products for gene doping assay validation in horses. Drug Test Anal 2022;14(6):1017‐1025.
- 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.
- Tozaki T, Ohnuma A, Kikuchi M. Simulated validation of intron‐less transgene detection using DELLY for gene‐doping control in horse sports. Anim Genet 2021;52(5):759‐761.
- Naumann N, Paßreiter A, Thomas A, Krug O, Walpurgis K, Thevis M. Analysis of potential gene doping preparations for transgenic DNA in the context of sports drug testing programs. Int J Mol Sci 2023;24(21):15835.
- Tozaki T, Ohnuma A, Kikuchi M. Investigation of optimal procedures for storage and use of plasma samples suitable for gene doping tests. J Equine Sci 2023;34(2):21‐27.
- Tozaki T, Ohnuma A, Kikuchi M. Robustness of digital PCR and real‐time PCR against inhibitors in transgene detection for gene doping control in equestrian sports. Drug Test Anal 2021;13(10):1768‐1775.
- Tozaki T, Ohnuma A, Iwai S. Robustness of digital PCR and real‐time PCR in transgene detection for gene‐doping control. Anal Chem 2021;93(18):7133‐7139.
Citations
This article has been cited 0 times.Use Nutrition Calculator
Check if your horse's diet meets their nutrition requirements with our easy-to-use tool Check your horse's diet with our easy-to-use tool
Talk to a Nutritionist
Discuss your horse's feeding plan with our experts over a free phone consultation Discuss your horse's diet over a phone consultation
Submit Diet Evaluation
Get a customized feeding plan for your horse formulated by our equine nutritionists Get a custom feeding plan formulated by our nutritionists
