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Drug testing and analysis2020; 13(1); 113-121; doi: 10.1002/dta.2907

A duplex qPCR assay for human erythropoietin (EPO) transgene to control gene doping in horses.

Abstract: The misuse of genetic manipulation technology to enhance athletic performance is termed gene doping which is prohibited in human sports, horseracing, and equestrian sports. Although many qPCR assays have been developed, most assays employ genomic DNA (gDNA) from humans, non-human primates, and mice as a background and they may not be applicable for testing horse samples. This study aimed to develop a qPCR assay for the detection of human erythropoietin (hEPO) transgene in horse blood cells where the viral vectors used in gene therapy can reside for months. For the detection of hEPO transgene, the performance of three sets of primers and a hydrolysis probe for hEPO were compared. One set showed adequate specificity, sensitivity, amplification efficiency, and a dynamic range of detection in the presence of horse gDNA. The assay was duplexed with the detection of horse tubulin α 4A (TUBA4A) gene as an endogenous internal control in order to prevent false-negative results due to poor recovery and storage of extracted DNA and/or qPCR experimental variation. For the extraction of hEPO-plasmid, the QIAGEN Gentra Puregene blood kit was shown to recover the majority (62%) of hEPO-plasmid from spiked horse blood cells. The specificity and limit of detection (LOD) of the duplex qPCR assay were determined in accordance with MIQE guidelines. These findings supported the application of this duplex qPCR assay to the detection of hEPO transgene in horse blood cells.
© 2020 John Wiley & Sons, Ltd.
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Publication Date: 2020-10-14 PubMed ID: 32762114DOI: 10.1002/dta.2907Google 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.

The researchers developed an effective duplex qPCR assay for detecting the human erythropoietin (hEPO) gene in horse blood cells, which could be used to prevent gene doping.

Understanding Key Concepts

  • Gene doping is a form of cheating in sports where athletes improve their performance by manipulating genetics, including use of the human erythropoietin (hEPO) gene. This gene has been known to enhance physical performance by promoting red blood cell production.
  • The technique being studied, duplex qPCR assay, is a method that can be used to detect specific genetic sequences – in this case, the hEPO gene. The ‘duplex’ refers to its ability to also measure a reference gene (in this case tubulin α 4A or TUBA4A) to ensure the test’s accuracy.

Research Methods

  • The research team tested three sets of primers and a hydrolysis probe for hEPO to compare their performance. Primers are used in qPCR assays to identify specific DNA sequences.
  • They selected the best-performing primer set based on specificity, sensitivity, amplification efficiency, and a dynamic range of detection in the presence of horse genomic DNA.
  • The duplex qPCR assay could detect both the hEPO transgene and horse TUBA4A gene. Including the TUBA4A gene allowed for an internal control to prevent false-negative results.
  • For the extraction of the hEPO-plasmid, researchers used the QIAGEN Gentra Puregene blood kit, which recovered the majority (62%) of hEPO-plasmid from spiked horse blood cells. The term ‘plasmid’ here refers to a small, circular piece of DNA that scientists insert into cells in gene doping.

Results

  • The specificity and limit of detection (LOD) of the duplex qPCR assay were determined according to the MIQE guidelines (Minimum Information for Publication of Quantitative Real-Time PCR Experiments) designed to ensure accuracy and reproducibility across different labs.
  • The results indicated that this duplex qPCR assay is precise and reliable for use in detecting the hEPO transgene in horse blood cells.

This research has significant implications for regulating human and equestrian sports and preventing fraudulent practices like gene doping.

Cite This Article

APA
Cheung HW, Wong KS, Lin VYC, Wan TSM, Ho ENM. (2020). A duplex qPCR assay for human erythropoietin (EPO) transgene to control gene doping in horses. Drug Test Anal, 13(1), 113-121. https://doi.org/10.1002/dta.2907

Publication

Drug testing and analysis
ISSN: 1942-7611
NlmUniqueID: 101483449
Country: England
Language: English
Volume: 13
Issue: 1
Pages: 113-121

Researcher Affiliations

Cheung, Hiu Wing
  • Racing Laboratory, The Hong Kong Jockey Club, Sha Tin Racecourse, Sha Tin, N.T., Hong Kong, China.
Wong, Kin-Sing
  • Racing Laboratory, The Hong Kong Jockey Club, Sha Tin Racecourse, Sha Tin, N.T., Hong Kong, China.
Lin, Venus Y C
  • Racing Laboratory, The Hong Kong Jockey Club, Sha Tin Racecourse, Sha Tin, N.T., Hong Kong, China.
Wan, Terence S M
  • Racing Laboratory, The Hong Kong Jockey Club, Sha Tin Racecourse, Sha Tin, N.T., Hong Kong, China.
Ho, Emmie N M
  • Racing Laboratory, The Hong Kong Jockey Club, Sha Tin Racecourse, Sha Tin, N.T., Hong Kong, China.

MeSH Terms

  • Animals
  • Blood Cells / metabolism
  • DNA / blood
  • DNA / genetics
  • Doping in Sports
  • Erythropoietin / genetics
  • Horses / blood
  • Horses / genetics
  • Humans
  • Real-Time Polymerase Chain Reaction / methods
  • Transgenes

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Citations

This article has been cited 8 times.
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