Investigation of optimal procedures for storage and use of plasma samples suitable for gene doping tests.
Abstract: Gene doping, which is prohibited in horseracing and equestrian sports, can be performed by introducing exogenous genes, known as transgenes, into the bodies of postnatal animals. To detect exogenous genes, a method utilizing quantitative polymerase chain reaction (qPCR) with a hydrolysis probe was developed to test whole blood and plasma samples, thereby protecting the fairness of competition and the rights of stakeholders in horseracing and equestrian sports. Therefore, we aimed to develop sample storage methods suitable for A and B samples in gene doping tests using blood. For sample A, sufficient qPCR detection was demonstrated after refrigeration for 1 to 2 weeks post collection. For sample B, the following procedures were confirmed to be suitable for storage: 1) centrifugation after sample receipt, 2) frozen storage, 3) natural thawing at room temperature, and 4) centrifugation without mixing blood cell components. Our results indicated that long-term cryopreservation yielded good plasma components from frozen blood samples even though it destroyed blood cells, indicating its applicability to the gene doping test using sample B, which can be stored for later use. Sample storage procedures are as important as detection methods in doping tests. Therefore, the series of procedures that we evaluated in this study will contribute to the efficient performance of gene doping tests through qPCR using blood samples.
©2023 The Japanese Society of Equine Science.
Publication Date: 2023-06-12 PubMed ID: 37405066PubMed Central: PMC10315635DOI: 10.1294/jes.34.21Google Scholar: Lookup
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Summary
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This research article discusses the optimal methods for storing and using plasma samples that are suitable for tests for gene doping in horseracing and equestrian sports.
Understanding Gene Doping
- Gene doping is an illegal practice that involves introducing exogenous genes or transgenes into postnatal animals, which earmarks it as a form of cheating in horseracing and equestrian sports.
- Researchers have used a method that employs quantitative polymerase chain reaction (qPCR) with a hydrolysis probe to detect these transgenes in blood and plasma samples.
Objectives of the Research
- The primary aim of this study was to develop effective storage methods for these blood samples in order to facilitate ongoing fairness in competition and protect the rights of the stakeholders in these sports.
- These samples, being categorized as ‘Sample A’ and ‘Sample B,’ needed to be handled using different procedures. Consequently, these specimens were dealt with in such a way that their storage would not impact the integrity of the qPCR testing.
Findings from the Study
- For sample A, refrigeration for 1 to 2 weeks post-collection was found to ensure sufficient qPCR detection.
- For sample B, researchers detailed the following suitable storage procedures: centrifugation upon receipt of sample, freezing the sample, allowing for natural thawing at room temperature, then centrifuging again without mixing the blood cell components.
The Role of Long-Term Cryopreservation
- It was discovered that long-term cryopreservation of blood samples, even though it destroys blood cells, still provides usable plasma components.
- This makes it a viable method for storing sample B, as it may need to be kept for future use.
Significance of Sample Storage Procedures
- Just as it matters how doping tests are conducted, it is also critically important how the samples used in these tests are stored. Poor storage can damage the blood cells and consequently might interfere with the qPCR testing.
- The research team therefore believes that the procedures found in this study to be effective will be a significant contribution to the ongoing efforts to reliably conduct gene doping tests using blood samples and qPCR.
Cite This Article
APA
Tozaki T, Ohnuma A, Kikuchi M, Ishige T, Kakoi H, Hirota KI, Takahashi Y, Nagata SI.
(2023).
Investigation of optimal procedures for storage and use of plasma samples suitable for gene doping tests.
J Equine Sci, 34(2), 21-27.
https://doi.org/10.1294/jes.34.21 Publication
Researcher Affiliations
- Genetic Analysis Department, Laboratory of Racing Chemistry, Tochigi 320-0851, Japan.
- Genetic Analysis Department, Laboratory of Racing Chemistry, Tochigi 320-0851, Japan.
- Genetic Analysis Department, Laboratory of Racing Chemistry, Tochigi 320-0851, Japan.
- Genetic Analysis Department, Laboratory of Racing Chemistry, Tochigi 320-0851, Japan.
- Genetic Analysis Department, Laboratory of Racing Chemistry, Tochigi 320-0851, Japan.
- Genetic Analysis Department, Laboratory of Racing Chemistry, Tochigi 320-0851, Japan.
- Equine Research Institute, Japan Racing Association, Tochigi 329-0412, Japan.
- Genetic Analysis Department, Laboratory of Racing Chemistry, Tochigi 320-0851, Japan.
References
This article includes 18 references
- Cheung HW, Wong KS, Lin VYC, Farrington AF, Bond AJ, Wan TSM, Ho ENM. Optimization and implementation of four duplex quantitative polymerase chain reaction assays for gene doping control in horseracing.. Drug Test Anal 2022 Sep;14(9):1587-1598.
- 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 Jan;13(1):113-121.
- Cywinska A, Szarska E, Kowalska A, Ostaszewski P, Schollenberger A. Gender differences in exercise--induced intravascular haemolysis during race training in thoroughbred horses.. Res Vet Sci 2011 Feb;90(1):133-7.
- Haughan J, Ortved KF, Robinson MA. Administration and detection of gene therapy in horses: A systematic review.. Drug Test Anal 2023 Feb;15(2):143-162.
- 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 Jun;12(6):743-751.
- 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 May;14(5):963-972.
- 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.
- Masini A, Tedeschi D, Baragli P, Sighieri C, Lubas G. Exercise-induced intravascular haemolysis in standardbred horses. Comp. Clin. Pathol. 2003;12:45–48.
- Okano M, Ikekita A, Sato M, Inoue T, Kageyama S, Akiyama K, Aoi A, Miyamoto A, Momobayashi A, Ota M, Ishige M, Sakurai H, Shiomura S, Takemine M, Watanabe Y, Hikota T. Doping control analyses during the Tokyo 2020 Olympic and Paralympic Games.. Drug Test Anal 2022 Nov;14(11-12):1836-1852.
- 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).
- Tozaki T, Hamilton NA. Control of gene doping in human and horse sports.. Gene Ther 2022 Apr;29(3-4):107-112.
- Tozaki T, Ohnuma A, Hamilton NA, Kikuchi M, Ishige T, Kakoi H, Hirota KI, Kusano K, Nagata SI. Low-copy transgene detection using nested digital polymerase chain reaction for gene-doping control.. Drug Test Anal 2022 Feb;14(2):382-387.
- Tozaki T, Ohnuma A, Iwai S, Kikuchi M, Ishige T, Kakoi H, Hirota K, Kusano K, Nagata S. Robustness of Digital PCR and Real-Time PCR in Transgene Detection for Gene-Doping Control.. Anal Chem 2021 May 11;93(18):7133-7139.
- 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).
- Tozaki T, Ohnuma A, Kikuchi M, Ishige T, Kakoi H, Hirota KI, Kusano K, Nagata SI. Robustness of digital PCR and real-time PCR against inhibitors in transgene detection for gene doping control in equestrian sports.. Drug Test Anal 2021 Oct;13(10):1768-1775.
- Tozaki T, Ohnuma A, Takasu M, Kikuchi M, Kakoi H, Hirota KI, Kusano K, Nagata SI. Droplet Digital PCR Detection of the Erythropoietin Transgene from Horse Plasma and Urine for Gene-Doping Control.. Genes (Basel) 2019 Mar 21;10(3).
- Tozaki T, Ohnuma A, Takasu M, Nakamura K, Kikuchi M, Ishige T, Kakoi H, Hirora KI, Tamura N, Kusano K, Nagata SI. Detection of non-targeted transgenes by whole-genome resequencing for gene-doping control.. Gene Ther 2021 Apr;28(3-4):199-205.
- 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.
Citations
This article has been cited 1 times.- 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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