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Genes2020; 11(4); 457; doi: 10.3390/genes11040457

Microfluidic Quantitative PCR Detection of 12 Transgenes from Horse Plasma for Gene Doping Control.

Abstract: Gene doping, an activity which abuses and misuses gene therapy, is a major concern in sports and horseracing industries. Effective methods capable of detecting and monitoring gene doping are urgently needed. Although several PCR-based methods that detect transgenes have been developed, many of them focus only on a single transgene. However, numerous genes associated with athletic ability may be potential gene-doping material. Here, we developed a detection method that targets multiple transgenes. We targeted 12 genes that may be associated with athletic performance and designed two TaqMan probe/primer sets for each one. A panel of 24 assays was prepared and detected via a microfluidic quantitative PCR (MFQPCR) system using integrated fluidic circuits (IFCs). The limit of detection of the panel was 6.25 copy/μL. Amplification-specificity was validated using several concentrations of reference materials and animal genomic DNA, leading to specific detection. In addition, target-specific detection was successfully achieved in a horse administered 20 mg of the transgene via MFQPCR. Therefore, MFQPCR may be considered a suitable method for multiple-target detection in gene-doping control. To our knowledge, this is the first application of microfluidic qPCR (MFQPCR) for gene-doping control in horseracing.
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Publication Date: 2020-04-23 PubMed ID: 32340130PubMed Central: PMC7230449DOI: 10.3390/genes11040457Google Scholar: Lookup
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  • 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.

The research proposes a new method for detecting gene doping in horses, focusing on detecting 12 transgenes related to athletic performance using a microfluidic quantitative PCR (MFQPCR) system. Traditional methods usually target only a single transgene.

Use of Microfluidic Quantitative PCR for Detection of Gene Doping

  • The researchers aim to address the need for effective detection of gene doping, which involves abusing gene therapy to enhance athletic ability, in particular, in horseracing.
  • Existing PCR-based methods typically target only one transgene, but with various genes potentially linked to athletic performance, the researchers decided to design a method that targets multiple transgenes.
  • They used a microfluidic quantitative PCR system, which enables rapid quantitative PCR on a tiny scale, to build a detection method for multiple gene targets.

Selection of Target Genes and Assay Preparation

  • Twelve genes which potentially influence athletic performance were selected as targets. Then, two TaqMan probe/primer sets were designed for each of these genes. TaqMan probe is a kind of oligonucleotide used in real-time PCR for DNA sequence detection.
  • A panel of 24 assays was created to carry out the detection on an integrated fluidic circuits (IFCs) platform. IFCs allow for precise handling and control of fluids in micro scaled volumes around the nanoliter scale. This technology is instrumental for microfluidic devices.

Test Sensitivity and Validity

  • The proposed detection panel can identify up to 6.25 copies/μL, providing a sensitive method of detection.
  • Multiple concentrations of reference materials and animal genomic DNA were used to validate the amplification-specificity of the test panel, leading to specific detection of each targeted gene.

Practical Application and Potential

  • The MFQPCR method successfully detected specific genes in a horse that was administrated 20 mg of the transgene. This suggests that this method can be applied in real-world scenarios for gene doping control amongst horses.
  • This is reportedly the first time MFQPCR has been used for gene doping control in horse racing, opening up new possibilities for the control and detection of gene doping in this field.

Cite This Article

APA
Tozaki T, Ohnuma A, Kikuchi M, Ishige T, Kakoi H, Hirota KI, Kusano K, Nagata SI. (2020). Microfluidic Quantitative PCR Detection of 12 Transgenes from Horse Plasma for Gene Doping Control. Genes (Basel), 11(4), 457. https://doi.org/10.3390/genes11040457

Publication

Genes
ISSN: 2073-4425
NlmUniqueID: 101551097
Country: Switzerland
Language: English
Volume: 11
Issue: 4
PII: 457

Researcher Affiliations

Tozaki, Teruaki
  • Genetic Analysis Department, Laboratory of Racing Chemistry, 1731-2 Tsurutamachi, Utsunomiya, Tochigi 320-0851, Japan.
Ohnuma, Aoi
  • Genetic Analysis Department, Laboratory of Racing Chemistry, 1731-2 Tsurutamachi, Utsunomiya, Tochigi 320-0851, Japan.
Kikuchi, Mio
  • Genetic Analysis Department, Laboratory of Racing Chemistry, 1731-2 Tsurutamachi, Utsunomiya, Tochigi 320-0851, Japan.
Ishige, Taichiro
  • Genetic Analysis Department, Laboratory of Racing Chemistry, 1731-2 Tsurutamachi, Utsunomiya, Tochigi 320-0851, Japan.
Kakoi, Hironaga
  • Genetic Analysis Department, Laboratory of Racing Chemistry, 1731-2 Tsurutamachi, Utsunomiya, Tochigi 320-0851, Japan.
Hirota, Kei-Ichi
  • Genetic Analysis Department, Laboratory of Racing Chemistry, 1731-2 Tsurutamachi, Utsunomiya, Tochigi 320-0851, Japan.
Kusano, Kanichi
  • Equine Department, Japan Racing Association, 6-11-1 Roppongi, Minato, Tokyo 106-8401, Japan.
Nagata, Shun-Ichi
  • Genetic Analysis Department, Laboratory of Racing Chemistry, 1731-2 Tsurutamachi, Utsunomiya, Tochigi 320-0851, Japan.

MeSH Terms

  • Animals
  • Athletic Performance
  • Doping in Sports / methods
  • Doping in Sports / prevention & control
  • Erythropoietin / blood
  • Erythropoietin / genetics
  • Horses
  • Male
  • Microfluidics / methods
  • Real-Time 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.

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Citations

This article has been cited 8 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.
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  2. Tozaki T, Ohnuma A, Kikuchi M, Ishige T, Kakoi H, Hirota KI, Takahashi Y, Nagata SI. Short Insertion and Deletion Discoveries via Whole-Genome Sequencing of 101 Thoroughbred Racehorses.. Genes (Basel) 2023 Mar 3;14(3).
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  3. 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
  4. 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.
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  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.
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