Digital PCR detection of plasmid DNA administered to the skeletal muscle of a microminipig: a model case study for gene doping detection.
Abstract: Doping control is an important and indispensable aspect of fair horse racing; genetic doping has been recently included to this. In this study, we aimed to develop a detection method of gene doping. A plasmid cloned with human erythropoietin gene (p.hEPO, 250 μg/head) was intramuscularly injected into a microminipig. Subsequently, p.hEPO was extracted from 1 mL of plasma and detected by droplet digital polymerase chain reaction. Results: The results confirmed that the maximum amount of plasmid was detected at 15 min after administration and the majority of the plasmid was degraded in the bloodstream within 1-2 days after administration. In contrast, low amounts of p.hEPO were detected at 2-3 weeks after administration. These results suggest that the proposed method to detect gene doping can help obtain information for experiments using horses.
Publication Date: 2018-10-10 PubMed ID: 30309394PubMed Central: PMC6180624DOI: 10.1186/s13104-018-3815-6Google Scholar: Lookup
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- Journal Article
Summary
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This study aimed to develop a method for detecting gene doping in horses using a plasmid cloned with human erythropoietin gene (p.hEPO) and tested on a microminipig. The results showed a possibility of detecting gene doping in the bloodstream for a period of 1-2 days after administration, with lower detection rates lasting up to 2-3 weeks.
Background of the Research
- Doping control is vital for ensuring fairness in sports such as horse racing.
- The focus of this research was gene doping, a relatively new form of cheating which includes altering the genetic makeup of athletes to enhance performance.
Objective of the Study
- The main aim was to develop a method for detecting gene doping.
- The researchers used a plasmid cloned with human erythropoietin gene (p.hEPO) in their method.
Methodology
- The p.hEPO (250 μg/head) was intramusically injected into a microminipig’s skeletal muscle.
- They then extracted the p.hEPO from a milliliter of plasma using the droplet digital polymerase chain reaction technique.
Findings of the Study
- The maximum amount of plasmid, according to the findings, could be detected about 15 minutes after it was administered.
- Most of the plasmid degraded in the bloodstream within 1-2 days of being administered, implying this would be the optimal time to detect gene doping.
- However, the research found that low amounts of p.hEPO could be detected 2-3 weeks after it was administered, signifying that detection remains possible for a longer duration, although the amounts diminish.
Implications of the Research
- The proposed method, as indicated by the findings of the research, could be employed in doping control for horse racing.
- This method could potentially help detect gene doping in the early stages post administration and could contribute to more accurate and fair sporting outcomes.
Cite This Article
APA
Tozaki T, Gamo S, Takasu M, Kikuchi M, Kakoi H, Hirota KI, Kusano K, Nagata SI.
(2018).
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, 11(1), 708.
https://doi.org/10.1186/s13104-018-3815-6 Publication
Researcher Affiliations
- 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.
- Department of Veterinary Medicine, Faculty of Applied Biological Sciences, Gifu University, 1-1 Yanagido, Gifu, Gifu, 501-1193, Japan.
- Department of Veterinary Medicine, Faculty of Applied Biological Sciences, Gifu University, 1-1 Yanagido, Gifu, Gifu, 501-1193, Japan.
- Genetic Analysis Department, Laboratory of Racing Chemistry, 1731-2 Tsurutamachi, Utsunomiya, Tochigi, 320-0851, Japan.
- Genetic Analysis Department, Laboratory of Racing Chemistry, 1731-2 Tsurutamachi, Utsunomiya, Tochigi, 320-0851, Japan.
- Genetic Analysis Department, Laboratory of Racing Chemistry, 1731-2 Tsurutamachi, Utsunomiya, Tochigi, 320-0851, Japan.
- Racehorse Hospital Ritto Training Center, Japan Racing Association, 1028 Misono, Ritto, Shiga, 520-3085, Japan.
- Genetic Analysis Department, Laboratory of Racing Chemistry, 1731-2 Tsurutamachi, Utsunomiya, Tochigi, 320-0851, Japan.
MeSH Terms
- Animals
- DNA Fragmentation
- Doping in Sports / methods
- Doping in Sports / prevention & control
- Erythropoietin / blood
- Erythropoietin / genetics
- Erythropoietin / pharmacokinetics
- Gene Expression
- Horses
- Humans
- Injections, Intramuscular
- Male
- Plasmids / administration & dosage
- Plasmids / chemistry
- Plasmids / metabolism
- Polymerase Chain Reaction / methods
- Recombinant Proteins / blood
- Recombinant Proteins / genetics
- Recombinant Proteins / pharmacokinetics
- Sensitivity and Specificity
- Swine
- Swine, Miniature
- Transgenes
Grant Funding
- 2017-2019 / JAPAN RACING ASSOCIATION
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Citations
This article has been cited 15 times.- 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).
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