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Drug testing and analysis2022; 14(6); 1017-1025; doi: 10.1002/dta.3219

Direct sequence confirmation of qPCR products for gene doping assay validation in horses.

Abstract: The misuse of gene therapy by the introduction of transgenes via plasmid or viral vectors as a doping agent is an increasing concern in human and animal sports, not only in consideration to fair competition but also in potential detrimental effects to welfare. Doping events can be detected by polymerase chain reaction (PCR) amplification of a transgene-specific region of DNA. Quantitative real-time PCR (qPCR) is particularly suited to confirmatory investigations where precise limits of detection can be calculated. To fully validate a qPCR experiment, it is highly desirable to confirm the identity of the amplicon. Although post-PCR techniques such as melt curve and fragment size analysis can provide strong evidence that the amplicon is as expected, sequence identity confirmation may be beneficial as part of regulatory proceedings. We present here our investigation into two alternative processes for the direct assessment of qPCR products for five genes using next-generation sequencing: ligation of sequence-ready adapters to qPCR products and qPCR assays performed with primers tailed with Illumina flow cell binding sites. To fully test the robustness of the techniques at concentrations required for gene doping detection, we also calculated a putative limit of detection for the assays. Both ligated adapters and tailed primers were successful in producing sequence data for the qPCR products without further amplification. Ligated adapters are preferred, however, as they do not require re-optimisation of existing qPCR assays.
© 2022 John Wiley & Sons, Ltd.
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Publication Date: 2022-01-20 PubMed ID: 34994083DOI: 10.1002/dta.3219Google 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.

The abstract discusses research on validating a method for detecting gene doping in horses through direct sequence confirmation of quantitative polymerase chain reaction (qPCR) products.

Overview of the Research

  • In the context of both human and animal sports, the abuse of gene therapy interventions, using either viral or plasmid vectors to administer transgenes, stands as an increasing problem. This gene doping not only raises issues of fairness in the competition, but could also threaten an athlete or animal’s well-being.
  • PCR (Polymerase Chain Reaction), a technique leveraged to amplify segments of DNA, can be used to detect instances of gene doping. Specifically, qPCR (Quantitative Real-Time PCR), a variant of PCR, is particularly useful for confirmation-based investigations as it can provide precise limits of detection. An integral part of qPCR validation involves confirming the identity of the amplified segment of DNA (amplicon).
  • While post-PCR techniques such as melt curve analysis and fragment size analysis can provide solid proof that the amplicon produced matches the expected result, sequence identity confirmation may provide beneficial contributions to regulatory procedures.

The Investigation and Findings

  • The researchers investigated two alternative processes for directly assessing qPCR products for five genes via next-generation sequencing:
    • The ligation of sequence-ready adapters to qPCR products
    • The performance of qPCR assays with primers tailed with sites that enable binding to an Illumina flow cell
  • The effectiveness of these techniques at the concentrations required for gene doping detection was tested by calculating a putative limit of detection for the assays.
  • Both techniques (ligated adapters and tailed primers) successfully produced sequence data for the qPCR products without needing further amplification. However, the research favored the use of ligated adapters since this method does not require existing qPCR assays to be re-optimized.

Cite This Article

APA
Maniego J, Pesko B, Hincks P, Taylor P, Stewart G, Proudman C, Scarth J, Ryder E. (2022). Direct sequence confirmation of qPCR products for gene doping assay validation in horses. Drug Test Anal, 14(6), 1017-1025. https://doi.org/10.1002/dta.3219

Publication

Drug testing and analysis
ISSN: 1942-7611
NlmUniqueID: 101483449
Country: England
Language: English
Volume: 14
Issue: 6
Pages: 1017-1025

Researcher Affiliations

Maniego, Jillian
  • Sport and Specialised Analytical Services, LGC, Cambridgeshire, UK.
Pesko, Bogusia
  • Sport and Specialised Analytical Services, LGC, Cambridgeshire, UK.
Hincks, Pamela
  • Sport and Specialised Analytical Services, LGC, Cambridgeshire, UK.
Taylor, Polly
  • Sport and Specialised Analytical Services, LGC, Cambridgeshire, UK.
Stewart, Graham
  • School of Biosciences and Medicine, University of Surrey, Guildford, UK.
Proudman, Christopher
  • School of Veterinary Medicine, University of Surrey, Guildford, UK.
Scarth, James
  • Sport and Specialised Analytical Services, LGC, Cambridgeshire, UK.
Ryder, Edward
  • Sport and Specialised Analytical Services, LGC, Cambridgeshire, UK.

MeSH Terms

  • Animals
  • DNA
  • DNA Primers
  • Doping in Sports
  • Horses
  • Real-Time Polymerase Chain Reaction / methods
  • Transgenes

Grant Funding

  • British Horseracing Authority

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Citations

This article has been cited 4 times.
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  3. Maniego J, Harding C, Habershon-Butcher J, Hincks P, Stewart G, Proudman C, Ryder E. Detection of transgenes in equine dried blood spots using digital PCR and qPCR for gene doping control. Drug Test Anal 2025 May;17(5):626-633.
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  4. Maniego J, Harding C, Habershon-Butcher J, Hincks P, Ryder E. Administration and detection of a multi-target rAAV gene doping vector in horses using multiple matrices and molecular techniques. Gene Ther 2024 Sep;31(9-10):477-488.
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