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Drug testing and analysis2021; 13(10); 1768-1775; doi: 10.1002/dta.3131

Robustness of digital PCR and real-time PCR against inhibitors in transgene detection for gene doping control in equestrian sports.

Abstract: Gene doping is a threat to fair competition in sports, both human and equestrian. One method of gene doping is to administer exogenous genetic materials, called transgenes, into the bodies of postnatal humans and horses. Polymerase chain reaction (PCR)-based transgene detection methods such as digital PCR and real-time PCR have been developed for gene doping testing in humans and horses. However, the significance of PCR inhibitors in gene doping testing has not been well evaluated. In this study, we evaluated the effects of PCR inhibitors on transgene detection using digital PCR and real-time PCR against gene doping. Digital PCR amplification was significantly inhibited by high concentrations of proteinase K (more than 0.1 μg/μl), ethylenediaminetetraacetic acid (more than 5 nmol/μl), and heparin (more than 0.05 unit/μl) but not by ethanol or genomic DNA. In addition, phenol affected droplet formation in the digital PCR amplification process. Real-time PCR amplification was inhibited by high concentrations of phenol (more than 1% v/v), proteinase K (more than 0.001 μg/μl), ethylenediaminetetraacetic acid (more than 1 nmol/μl), heparin (more than 0.005 unit/μl), and genomic DNA (more than 51.9 ng/μl) but not by ethanol. Although both PCR systems were inhibited by nearly the same substances, digital PCR was more robust than real-time PCR against the inhibitors. We believe that our findings are important for the development of better methods for transgene detection and prevention of false negative results in gene doping testing.
© 2021 John Wiley & Sons, Ltd.
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Publication Date: 2021-07-21 PubMed ID: 34270866DOI: 10.1002/dta.3131Google Scholar: Lookup
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Summary

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This study researches the efficacy and resistance of digital PCR and real-time PCR transgene detection methods against certain inhibitors, in order to improve testing for gene doping in equestrian sports. The research found that while both types of PCR were impeded by close to identical substances, digital PCR was more resilient.

Analysis of PCR-based Transgene Detection Techniques

  • The goal of the research was to enhance the understanding of gene doping detection, which uses exogenous genetic materials called transgenes.
  • Gene doping is a concern in both human and equestrian sports, compromising the fairness of competition.
  • This study focuses on Polymerase chain reaction (PCR)-based transgene detection methods such as digital PCR and real-time PCR. PCR is a widely-used molecular biology technique for amplifying DNA to detectable levels.
  • Specifically, it evaluates the influence of PCR inhibitors on transgene detection capacity, addressing a gap in prior research that insufficiently examined the significance of such inhibitors in gene doping testing.

Influence of Various PCR Inhibitors

  • Different inhibitors were tested on digital PCR and real-time PCR for their effects. Inhibition signifies a reduction in the effectiveness of PCR reaction, which can produce false negative results in gene doping tests – indicating the absence of doping when it is actually present.
  • Inhibitors tested include high concentrations of proteinase K, ethylenediaminetetraacetic acid (EDTA), heparin, ethanol, genomic DNA, and phenol.
  • Digital PCR amplification was significantly impeded by proteinase K, EDTA, and heparin, but was unaffected by ethanol or genomic DNA. Phenol disrupted droplet formation in the digital PCR amplification process.

Comparison of Digital PCR vs Real-Time PCR

  • Real-time PCR amplification was inhibited by phenol, proteinase K, EDTA, heparin, and genomic DNA, similar to the substances that impeded digital PCR. However, real-time PCR was not affected by ethanol.
  • In comparison, digital PCR proved more resistant or robust against the tested inhibitors than real-time PCR, particularly notable with respect to genomic DNA, an inhibitor that affected real-time PCR but not digital PCR.
  • The researchers suggest that this makes digital PCR potentially more reliable for gene doping testing, as it can provide accurate results despite the presence of inhibitors.

Implications for Gene Doping Detection

  • The results of this study provide vital information for improving transgene detection methods and preventing false negatives in gene doping testing.
  • By understanding the robustness of the PCR systems against certain inhibitors, scientists can develop better testing methods to maintain fair competition within equestrian and potentially other sports.

Cite This Article

APA
Tozaki T, Ohnuma A, Kikuchi M, Ishige T, Kakoi H, Hirota KI, Kusano K, Nagata SI. (2021). Robustness of digital PCR and real-time PCR against inhibitors in transgene detection for gene doping control in equestrian sports. Drug Test Anal, 13(10), 1768-1775. https://doi.org/10.1002/dta.3131

Publication

Drug testing and analysis
ISSN: 1942-7611
NlmUniqueID: 101483449
Country: England
Language: English
Volume: 13
Issue: 10
Pages: 1768-1775

Researcher Affiliations

Tozaki, Teruaki
  • Genetic Analysis Department, Laboratory of Racing Chemistry, Utsunomiya, Tochigi, Japan.
Ohnuma, Aoi
  • Genetic Analysis Department, Laboratory of Racing Chemistry, Utsunomiya, Tochigi, Japan.
Kikuchi, Mio
  • Genetic Analysis Department, Laboratory of Racing Chemistry, Utsunomiya, Tochigi, Japan.
Ishige, Taichiro
  • Genetic Analysis Department, Laboratory of Racing Chemistry, Utsunomiya, Tochigi, Japan.
Kakoi, Hironaga
  • Genetic Analysis Department, Laboratory of Racing Chemistry, Utsunomiya, Tochigi, Japan.
Hirota, Kei-Ichi
  • Genetic Analysis Department, Laboratory of Racing Chemistry, Utsunomiya, Tochigi, Japan.
Kusano, Kanichi
  • Equine Department, Japan Racing Association, Minato, Tokyo, Japan.
Nagata, Shun-Ichi
  • Genetic Analysis Department, Laboratory of Racing Chemistry, Utsunomiya, Tochigi, Japan.

MeSH Terms

  • Animals
  • Doping in Sports / prevention & control
  • Horses
  • Humans
  • Polymerase Chain Reaction / methods
  • Polymerase Chain Reaction / veterinary
  • Real-Time Polymerase Chain Reaction / veterinary
  • Transgenes

Grant Funding

  • Japan Racing Association

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Citations

This article has been cited 3 times.
  1. Lu Y, Yan J, Ou G, Fu L. A Review of Recent Progress in Drug Doping and Gene Doping Control Analysis.. Molecules 2023 Jul 18;28(14).
    doi: 10.3390/molecules28145483pubmed: 37513354google scholar: lookup
  2. 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
  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
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