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Drug testing and analysis2021; 14(2); 382-387; doi: 10.1002/dta.3173

Low-copy transgene detection using nested digital polymerase chain reaction for gene-doping control.

Abstract: Gene doping is prohibited for fair competition in human and horse sports. One style of gene doping is the administration of an exogeneous gene, called a transgene, to postnatal humans and horses. Although many transgene detection methods based on quantitative polymerase chain reaction (PCR), including real-time PCR and digital PCR, have been recently developed, it remains difficult to reliably detect low-copy transgenes. In this study, we developed and validated a nested digital PCR method to specifically detect low-copy transgenes. The nested digital PCR consists of (1) preamplification using conventional PCR and (2) droplet digital PCR detection using a hydrolysis probe. Using 5, 10, 20, 60 and 120 transgene copies as template, 496.0, 1089.7, 1820.7, 4313.3 and 7840.0 copies per microlitre, respectively, were detected using our nested digital PCR. Although high concentrations of phenol, proteinase K, ethanol, EDTA, heparin and genomic DNA all inhibited preamplification, their effects on the digital PCR detection were limited. Once preamplification was successful, even substitution of bases within the primers and probes had minimal effects on transgene detection. The nested digital PCR developed in this study successfully detected low-copy transgenes and can be used to perform a qualitative test, indicating its usefulness in the prevention of false positives and false negatives in gene-doping detection.
© 2021 John Wiley & Sons, Ltd.
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Publication Date: 2021-10-18 PubMed ID: 34608764DOI: 10.1002/dta.3173Google Scholar: Lookup
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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.

This study presents a new method for detecting low-copy transgenes – genetic material modified by gene doping – by using nested digital polymerase chain reaction (PCR). The researchers found that the method could detect small amounts of transgenes even in the presence of substances that traditionally inhibit detection.

Introduction to Gene Doping

  • The paper starts with a discussion on gene doping, a banned practice in human and horse sports that involves the administration of an exogeneous gene, referred to as a transgene, to postnatal humans and horses for performance enhancement.

Issues with Existing Detection Methods

  • The authors point to the difficulty of detecting low-copy transgenes with existing transgene detection methods based on quantitative polymerase chain reaction (PCR), including real-time PCR and digital PCR.

Development of Nested Digital PCR

  • To overcome these challenges, the researchers developed a nested digital PCR method.
  • This method includes two parts: preamplification using conventional PCR and droplet digital PCR detection using a hydrolysis probe.
  • The authors present the results from using 5, 10, 20, 60, and 120 transgene copies as template, which resulted in the detection of 496.0, 1089.7, 1820.7, 4313.3, and 7840.0 copies per microlitre, respectively.

Resistance to Inhibition and Accuracy

  • This new method was found to be resilient to high concentrations of substances that may otherwise inhibit preamplification, such as phenol, proteinase K, ethanol, EDTA, heparin, and genomic DNA.
  • The effects of these inhibitors on the digital PCR detection were minimal, and successful preamplification could also withstand some level of base substitution within the primers and probes without affecting transgene detection significantly.

Implication for Gene Doping Control

  • The researchers argue that their nested digital PCR method can effectively detect low-copy transgenes, making it a valuable tool for performing a qualitative test.
  • It would be beneficial in preventing false positives and false negatives in gene doping detections, and therefore, contribute to maintaining fair competition in sports.

Cite This Article

APA
Tozaki T, Ohnuma A, Hamilton NA, Kikuchi M, Ishige T, Kakoi H, Hirota KI, Kusano K, Nagata SI. (2021). Low-copy transgene detection using nested digital polymerase chain reaction for gene-doping control. Drug Test Anal, 14(2), 382-387. https://doi.org/10.1002/dta.3173

Publication

Drug testing and analysis
ISSN: 1942-7611
NlmUniqueID: 101483449
Country: England
Language: English
Volume: 14
Issue: 2
Pages: 382-387

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.
Hamilton, Natasha A
  • Equine Genetics Research Centre, Racing Australia, Scone, New South Wales, Australia.
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
  • DNA / genetics
  • DNA Primers
  • Doping in Sports / methods
  • Doping in Sports / prevention & control
  • Horses / genetics
  • Real-Time Polymerase Chain Reaction / methods
  • Transgenes

Grant Funding

  • Japan Racing Association

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This article includes 26 references
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Citations

This article has been cited 4 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
  4. Thomas A, Walpurgis K, Naumann N, Piper T, Thevis M. Bioanalytical methods in doping controls: a review. Bioanalysis 2025 Mar;17(5):359-370.
    doi: 10.1080/17576180.2025.2460951pubmed: 39916648google scholar: lookup
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