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Home / Equine Research Bank / Drug Test Anal / Optimization and implementation of four duplex quantitative ...
Drug testing and analysis2022; 14(9); 1587-1598; doi: 10.1002/dta.3328

Optimization and implementation of four duplex quantitative polymerase chain reaction assays for gene doping control in horseracing.

Abstract: The concern about gene doping has remained high in horseracing and other equestrian competitions. Our laboratory has previously developed a duplex quantitative polymerase chain reaction (qPCR) assay capable of detecting in equine blood the human erythropoietin (hEPO) transgene and equine tubulin α 4a (TUBA4A) gene as an internal control the latter providing quality control over DNA extraction and qPCR. This study aimed to optimize the method for routine testing of regulatory samples. The use of an automated DNA extraction system has increased the sample throughput, consistency of DNA extraction, and recovery of reference materials. The use of reduced concentration of primers and hydrolysis probe for internal control minimized their competition with transgene amplification and improved the assay sensitivity. Spike-in of an exogenous internal control at low concentration for plasma analysis has also been validated. Using the new workflow, four duplex qPCR assays have been developed for the detection of transgenes, namely, hEPO, human growth hormone (hGH), insulin-like growth factor 1 (hIGF-1), and equine EPO (eEPO). The estimated limits of detection (LODs) of each transgene were 2000 copies/mL of blood and 200 copies/mL of plasma. This method could detect the presence of transgene in blood and plasma collected from a horse administered intramuscularly (IM) with recombinant adeno-associated virus (rAAV) carrying the hEPO transgene. A longer detection time was observed in blood than in plasma. The methods have been applied to the screening of over a thousand official racehorse samples since June 2020 for the presence of these transgenes.
© 2022 John Wiley & Sons Ltd.
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Publication Date: 2022-06-06 PubMed ID: 35633307DOI: 10.1002/dta.3328Google 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.

This research focused on improving methods for detecting gene doping in horserace horses through the development of an enhanced real-time polymerase chain reaction (qPCR) testing mechanism. The refined technique, which was applied to over a thousand official racehorse samples, resulted in the successful detection of specific illicit transgenes in blood and plasma.

Introduction

  • The research focuses on gene doping, a prevalent concern in horseracing and other equestrian sports.
  • The authors previously created a duplex qPCR test that could identify the human erythropoietin (hEPO) transgene and equine tubulin α 4a (TUBA4A) gene in horse blood. The TUBA4A gene acted as an internal control, providing quality assurance for DNA extraction and qPCR.
  • This latest study worked to enhance this testing method for routine usage in regulatory sample checking.

Methods

  • The researchers used an automated DNA extraction system to increase the efficacy and consistency of DNA extraction, and to improve the recovery of reference materials.
  • They also reduced the concentration of primers and the hydrolysis probe for the internal control to lessen their competition with transgene amplification, which allowed for greater test sensitivity.
  • The study validated the spike-in of a low concentration exogenous internal control for plasma analysis.

Results

  • Following the new workflow, four duplex qPCR tests were designed for the detection of the following transgenes: hEPO, human growth hormone (hGH), insulin-like growth factor 1 (hIGF-1), and equine EPO (eEPO).
  • The estimated limits of detection (LODs) of each transgene were 2000 copies/mL in blood and 200 copies/mL in plasma.
  • The refined method was successfully able to identify the presence of the hEPO transgene in blood and plasma from a horse that had been intramuscularly administered with a recombinant adeno-associated virus (rAAV) carrying the hEPO transgene. It was also noted that the detection time was longer in blood than in plasma.

Conclusion

  • Since June 2020, the enhanced methods have been applied in the screening of over a thousand racehorse samples for the presence of these transgenes.

Cite This Article

APA
Cheung HW, Wong KS, Lin VYC, Farrington AF, Bond AJ, Wan TSM, Ho ENM. (2022). Optimization and implementation of four duplex quantitative polymerase chain reaction assays for gene doping control in horseracing. Drug Test Anal, 14(9), 1587-1598. https://doi.org/10.1002/dta.3328

Publication

Drug testing and analysis
ISSN: 1942-7611
NlmUniqueID: 101483449
Country: England
Language: English
Volume: 14
Issue: 9
Pages: 1587-1598

Researcher Affiliations

Cheung, Hiu Wing
  • Racing Laboratory, The Hong Kong Jockey Club, Sha Tin Racecourse, Sha Tin, Hong Kong, China.
Wong, Kin-Sing
  • Racing Laboratory, The Hong Kong Jockey Club, Sha Tin Racecourse, Sha Tin, Hong Kong, China.
Lin, Venus Y C
  • Racing Laboratory, The Hong Kong Jockey Club, Sha Tin Racecourse, Sha Tin, Hong Kong, China.
Farrington, Adrian F
  • Veterinary Clinical Services, The Hong Kong Jockey Club, Sha Tin Racecourse, Sha Tin, Hong Kong, China.
Bond, Amanda J
  • Equestrian Affairs, The Hong Kong Jockey Club, Sha Tin Racecourse, Sha Tin, Hong Kong, China.
Wan, Terence S M
  • Racing Laboratory, The Hong Kong Jockey Club, Sha Tin Racecourse, Sha Tin, Hong Kong, China.
Ho, Emmie N M
  • Racing Laboratory, The Hong Kong Jockey Club, Sha Tin Racecourse, Sha Tin, Hong Kong, China.

MeSH Terms

  • Animals
  • DNA
  • DNA Primers
  • Dependovirus / genetics
  • Doping in Sports / prevention & control
  • Horses / genetics
  • Humans
  • Polymerase Chain Reaction / methods
  • Recombinant Proteins
  • Transgenes

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Citations

This article has been cited 2 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, 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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