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Home / Equine Research Bank / Drug Test Anal / Detection of transgenes in equine dried blood spots using di...
Drug testing and analysis2024; 17(5); 626-633; doi: 10.1002/dta.3755

Detection of transgenes in equine dried blood spots using digital PCR and qPCR for gene doping control.

Abstract: Due to the ease of collection, transport and storage, the use of dried blood spots (DBS) offers an attractive alternative matrix for detection of the abuse of gene therapy, otherwise known as gene doping. This study evaluated the recovery, extraction efficiency and resulting detection capability of DNA from DBS by evaluating different target types, DNA extraction kits, the number of punches and blood tube preservatives. The long-term storage stability of low-copy-number transgene targets in DBS was not assessed in this study but would be noteworthy to investigate further. DNA was quantified using two detection methods: qPCR and digital PCR (dPCR). Using six punches with the Qiagen Investigator kit gave the best overall DNA yield compared with other extraction methods. Including three punches, however, gave better DNA extraction efficiency. Reference material could be detected using qPCR and dPCR in DBS spiked with 5000 copies/mL of blood (approximately 15 copies per 3 mm of punch). The optimal DNA extraction protocol was used on DBS samples from a custom recombinant adeno-associated virus administration study and showed successful detection of vector targets in DBS samples.
© 2024 British Horseracing Authority and LGC Ltd. Drug Testing and Analysis published by John Wiley & Sons Ltd.
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Publication Date: 2024-07-11 PubMed ID: 38992991PubMed Central: PMC12012414DOI: 10.1002/dta.3755Google Scholar: Lookup
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  • Journal Article

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.

Overview

  • This research investigates the effectiveness of using dried blood spots (DBS) combined with digital PCR (dPCR) and quantitative PCR (qPCR) to detect gene doping by identifying transgenes in horses.

Background and Purpose

  • Gene doping, involving the abuse of gene therapy to enhance performance in horses, requires reliable detection methods.
  • Dried blood spots (DBS) are considered an attractive sample matrix because they are easy to collect, transport, and store.
  • The study’s main goal is to evaluate multiple factors that affect DNA recovery and amplification from DBS for detecting transgenes.

Methodology

  • Sample Preparation:
    • DBS samples were prepared, including those spiked with known quantities of transgenic DNA (reference material) to mimic gene doping.
    • A custom recombinant adeno-associated virus (AAV) was administered in a separate study to generate real DBS samples with vector targets.
  • DNA Extraction:
    • Various DNA extraction kits were tested to compare DNA yield and quality.
    • The study varied the number of DBS punches used for extraction, testing both three and six punches from a 3 mm diameter spot.
    • Blood tube preservatives were also evaluated to determine their impact on DNA recovery.
  • Detection Methods:
    • Quantitative PCR (qPCR) and digital PCR (dPCR) were employed to quantify and detect the transgene DNA.
    • These two PCR methods were compared for sensitivity and reliability in detecting low-copy-number targets.

Key Findings

  • DNA Yield:
    • Using six punches with the Qiagen Investigator kit achieved the highest overall DNA yield.
    • However, using only three punches produced better DNA extraction efficiency, meaning more DNA per unit of input material was recovered.
  • Detection Sensitivity:
    • Both qPCR and dPCR successfully detected transgene reference material in DBS samples spiked with approximately 5000 copies/mL of blood.
    • This concentration corresponded roughly to 15 copies per 3 mm punch, showing the methods’ capability to detect low levels of transgenic DNA.
  • Application on Real Samples:
    • The optimal DNA extraction protocol was applied to DBS samples from the recombinant AAV administration study.
    • Detection of vector DNA targets in these real DBS samples was successful, demonstrating practical application potential.
  • Limitations:
    • The study did not assess the long-term storage stability of low-copy-number transgene targets in DBS, an important consideration for real-world testing scenarios.
    • Further research was suggested to evaluate how DNA degradation or stability might affect gene doping detection over time.

Significance and Implications

  • This study supports the use of DBS as a minimally invasive and practical sample type for gene doping control in horses.
  • It highlights that both dPCR and qPCR are effective detection tools for low-abundance transgenes in DBS.
  • The findings provide a foundation to develop robust protocols for sample collection and DNA extraction to enhance doping surveillance.
  • Understanding DNA extraction efficiencies and detection limits will assist labs in standardizing gene doping tests to ensure sensitivity and accuracy.
  • Future work on storage stability will further validate the practicality of DBS for longitudinal doping control programs.

Cite This Article

APA
Maniego J, Harding C, Habershon-Butcher J, Hincks P, Stewart G, Proudman C, Ryder E. (2024). Detection of transgenes in equine dried blood spots using digital PCR and qPCR for gene doping control. Drug Test Anal, 17(5), 626-633. https://doi.org/10.1002/dta.3755

Publication

Drug testing and analysis
ISSN: 1942-7611
NlmUniqueID: 101483449
Country: England
Language: English
Volume: 17
Issue: 5
Pages: 626-633

Researcher Affiliations

Maniego, Jillian
  • Sport and Specialised Analytical Services, LGC, Fordham, UK.
Harding, Caitlin
  • Sport and Specialised Analytical Services, LGC, Fordham, UK.
Habershon-Butcher, Jocelyn
  • British Horseracing Authority, London, UK.
Hincks, Pamela
  • Sport and Specialised Analytical Services, LGC, Fordham, UK.
Stewart, Graham
  • School of Biosciences and Medicine, University of Surrey, Guildford, UK.
Proudman, Christopher
  • School of Veterinary Medicine, University of Surrey, Guildford, UK.
Ryder, Edward
  • Sport and Specialised Analytical Services, LGC, Fordham, UK.

MeSH Terms

  • Animals
  • Dried Blood Spot Testing / methods
  • Dried Blood Spot Testing / veterinary
  • Doping in Sports
  • Horses / blood
  • Horses / genetics
  • Real-Time Polymerase Chain Reaction / methods
  • Transgenes / genetics
  • DNA / blood
  • DNA / genetics
  • Dependovirus / genetics
  • Genetic Therapy
  • Polymerase Chain Reaction / methods

Grant Funding

  • British Horseracing Authority (BHA)

Conflict of Interest Statement

The authors declare no conflict of interest.

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Citations

This article has been cited 2 times.
  1. Marchand A, Roulland I, Ericsson M. Improvement of EPO Transgene Detection From Polymeric Dried Blood Spots for Antidoping Application. Drug Test Anal 2026 Feb;18(2):230-238.
    doi: 10.1002/dta.70008pubmed: 41308298google scholar: lookup
  2. Han J, Ganguly R, Yi JY, Yun H, Jung SY, Sung C, Lee CS. Osmotically Tunable Microdroplets Enable Amplification-Free CRISPR Detection of Gene Doping. Adv Sci (Weinh) 2025 Dec;12(48):e15861.
    doi: 10.1002/advs.202515861pubmed: 41084992google scholar: lookup
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