FREE SHIPPING over $40
OVER 9000 FIVE-STAR REVIEWS
5% OFF ALL SUBSCRIPTIONS
100% HAPPINESS GUARANTEE
Analyze Diet
0
Home / Equine Research Bank / Drug Test Anal / A multiplex qPCR assay for transgenes detection: A novel app...
Drug testing and analysis2023; 15(8); 879-888; doi: 10.1002/dta.3483

A multiplex qPCR assay for transgenes detection: A novel approach for gene doping control in horseracing using conventional laboratory setup.

Abstract: Illicit administration of transgene into horses is a form of gene doping that has been a key concern in equine sports. The large number of potential performance-enhancing transgenes has demanded a cost-effective and reliable detection method. Multiplex qPCR is a relevant technique, but the cross-talking between fluorophores and high background noise limits the method sensitivity and specificity. This study reports a simpler multiplexing approach by using the same fluorophore for four hydrolysis probes each targeting one of the four transgenes: human growth hormone, insulin-like growth factor 1, equine erythropoietin and interleukin-10. Any positive findings from this multiplex qPCR assay can then be confirmed by individual qPCR assays to identify potential transgene(s). This has effectively eliminated the cross-talking issue and allowed an improved signal-to-noise than conventional multiplex qPCR assay. It has also removed the limitation imposed by the available choice of fluorophores and optical channels of qPCR instruments on the number of transgenes that can be analysed in a multiplex qPCR assay. This novel multiplex qPCR has been successfully validated. The estimated limits of detection were ~1500-2500 copies/mL of blood, thus demonstrating comparable sensitivity with the corresponding duplex qPCR assays. Concurring results were obtained by analysing hundreds of official blood samples provided by racehorses with this multiplex qPCR assay and the accredited individual duplex qPCR assays. This novel multiplex qPCR assay for detecting multiple transgenes is a cost-effective screening method using a conventional laboratory setup and has opened up the potential to include the testing of additional transgenes in a single assay.
© 2023 John Wiley & Sons Ltd.
Get Full Text
Publication Date: 2023-04-19 PubMed ID: 37056164DOI: 10.1002/dta.3483Google Scholar: Lookup
The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
LinkedInCopy
  • 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 research article presents a novel multiplex qPCR assay technique for the detection of illicit administration of performance-enhancing transgenes in horses. This is a more cost-effective and reliable method that overcomes the limitations of conventional multiplex qPCR and can serve as a significant tool in equine sports doping control.

Objective of the Study

  • The major objective of this study was to devise a new assay technique for detecting gene doping in competitive horseracing. The illicit administration of transgenes to enhance performance is a growing concern in the industry. Hence, a sensitive, reliable, and cost-effective detection method was needed.

Methodology

  • The researchers proposed a simpler multiplexing approach by using the same fluorophore for four different hydrolysis probes. Each of these probes targets a specific transgene such as human growth hormone, insulin-like growth factor 1, equine erythropoietin, and interleukin-10.
  • The detection was followed by individual qPCR assays to confirm any positive findings. Consequently, the cross-talking issue, a major limitation of the traditional multiplex qPCR, was addressed allowing an improved signal-to-noise ratio.
  • This technique also overcame the limitations imposed by the available choice of fluorophores and the optical channels of qPCR instruments on the number of transgenes that could be analysed in a multiplex qPCR assay.

Result and Findings

  • The proposed method was validated successfully. The estimated detection limits were around 1,500 to 2,500 copies/mL of blood, providing a sensitivity level that is comparable with corresponding duplex qPCR assays.
  • The newly devised technique was tested against a large number of official blood samples from racehorses and found to produce consistent results with the accredited individual duplex qPCR assays.
  • Overall, the novel multiplex qPCR assay was effective in detecting multiple transgenes in a single test using a conventional laboratory setup.

Significance of the Study

  • This study provides a novel, cost-effective screening method for detecting multiple transgenes in horse doping, adding a valuable tool in the field of equine sports doping control. This can also open up potential for inclusive testing of additional transgenes in a single assay.

Cite This Article

APA
Wong KS, Cheung HW, Szeto CWL, Tsang CYN, Wan TSM, Ho ENM. (2023). A multiplex qPCR assay for transgenes detection: A novel approach for gene doping control in horseracing using conventional laboratory setup. Drug Test Anal, 15(8), 879-888. https://doi.org/10.1002/dta.3483

Publication

Drug testing and analysis
ISSN: 1942-7611
NlmUniqueID: 101483449
Country: England
Language: English
Volume: 15
Issue: 8
Pages: 879-888

Researcher Affiliations

Wong, Kin-Sing
  • Racing Laboratory, The Hong Kong Jockey Club, Sha Tin Racecourse, Sha Tin, N.T., Hong Kong, China.
Cheung, Hiu Wing
  • Racing Laboratory, The Hong Kong Jockey Club, Sha Tin Racecourse, Sha Tin, N.T., Hong Kong, China.
Szeto, Cherry W L
  • Racing Laboratory, The Hong Kong Jockey Club, Sha Tin Racecourse, Sha Tin, N.T., Hong Kong, China.
Tsang, Candice Y N
  • 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

  • Humans
  • Animals
  • Horses / genetics
  • Doping in Sports / prevention & control
  • Transgenes
  • Erythropoietin / genetics
  • Sensitivity and Specificity
  • Real-Time Polymerase Chain Reaction / methods

References

This article includes 37 references
  1. Unal M, Ozer UD. Gene doping in sports. Sports Med 2004;34(6):357-362.
    doi: 10.2165/00007256-200434060-00002google scholar: lookup
  2. International Federation of Horseracing Authorities. Article 6 of the International Agreement on Breeding, Racing and Wagering and Appendixes. Accessed January 18, 2023. https://www.ifhaonline.org/default.asp?section=IABRW&AREA=2#article6
  3. Fédération Équestre Internationale. FEI Veterinary Regulations 2023. https://inside.fei.org/content/fei-veterinary-regulations-2023-0. Accessed January 18, 2023.
  4. Brzeziańska E, Domańska D, Jegier A. Gene doping in sport-perspectives and risks. Biol Sport 2014;31(4):251-259.
    doi: 10.5604/20831862.1120931google scholar: lookup
  5. Li C, Samulski RJ. Engineering adeno-associated virus vectors for gene therapy. Nat Rev Genet 2020;21(4):255-272.
    doi: 10.1038/s41576-019-0205-4google scholar: lookup
  6. Paßreiter A, Thomas A, Grogna N, Delahaut P, Thevis M. First steps toward uncovering gene doping with CRISPR/Cas by identifying SpCas9 in plasma via HPLC- HRMS/MS. Anal Chem 2020;92(24):16322-16328.
    doi: 10.1021/acs.analchem.0c04445google scholar: lookup
  7. Frisbie DD, Ghivizzani SC, Robbins PD, Evans CH, McIlwraith CW. Treatment of experimental equine osteoarthritis by in vivo delivery of the equine interleukin-1 receptor antagonist gene. Gene Ther 2002;9(1):12-20.
    doi: 10.1038/sj.gt.3301608google scholar: lookup
  8. Hemphill DD, McIlwraith CW, Slayden RA, Samulski RJ, Goodrich LR. Adeno-associated virus gene therapy vector scAAVIGF-I for transduction of equine articular chondrocytes and RNA-seq analysis. Osteoarthr Cartil 2016;24(5):902-911.
    doi: 10.1016/j.joca.2015.12.001google scholar: lookup
  9. Kovac M, Litvin YA, Aliev RO. Gene therapy using plasmid DNA encoding vascular endothelial growth factor 164 and fibroblast growth factor 2 genes for the treatment of horse tendinitis and desmitis: case reports. Front Vet Sci 2017;4:168.
    doi: 10.3389/fvets.2017.00168google scholar: lookup
  10. Moss KL, Jiang Z, Dodson ME. Sustained interleukin-10 transgene expression following intra-articular AAV5-IL-10 administration to horses. Hum Gene Ther 2020;31(1-2):110-118.
    doi: 10.1089/hum.2019.195google scholar: lookup
  11. Watson Levings RS, Broome TA, Smith AD. Gene therapy for osteoarthritis: pharmacokinetics of intra-articular self-complementary adeno-associated virus interleukin-1 receptor antagonist delivery in an equine model. Hum Gene Ther Clin Dev 2018;29(2):90-100.
    doi: 10.1089/humc.2017.142google scholar: lookup
  12. Watson Levings RS, Smith AD, Broome TA. Self-complementary adeno-associated virus-mediated Interleukin-1 receptor antagonist gene delivery for the treatment of osteoarthritis: test of efficacy in an equine model. Hum Gene Ther Clin Dev 2018;29(2):101-112.
    doi: 10.1089/humc.2017.143google scholar: lookup
  13. Cheung HW, Wong KS, Lin VYC. Optimization and implementation of four duplex quantitative polymerase chain reaction assays for gene doping control in horseracing. Drug Test Anal 2022;14(9):1587-1598.
    doi: 10.1002/dta.3328google scholar: lookup
  14. Haughan J, Jiang Z, Stefanovski D, Moss KL, Ortved KF, Robinson MA. Detection of intra-articular gene therapy in horses using quantitative real time PCR in synovial fluid and plasma. Drug Test Anal 2020;12(6):743-751.
    doi: 10.1002/dta.2785google scholar: lookup
  15. Cheung HW, Wong KS, Lin VYC, Wan TSM, Ho ENM. A duplex qPCR assay for human erythropoietin (EPO) transgene to control gene doping in horses. Drug Test Anal 2021;13(1):113-121.
    doi: 10.1002/dta.2907google scholar: lookup
  16. Jiang Z, Haughan J, Moss KL, Stefanovski D, Ortved KF, Robinson MA. A quantitative PCR screening method for adeno-associated viral vector 2-mediated gene doping. Drug Test Anal 2022;14(5):963-972.
    doi: 10.1002/dta.3152google scholar: lookup
  17. Maniego J, Pesko B, Habershon-Butcher J. Screening for gene doping transgenes in horses via the use of massively parallel sequencing. Gene Ther 2022;29(5):236-246.
    doi: 10.1038/s41434-021-00279-1google scholar: lookup
  18. Tozaki T, Ohnuma A, Takasu M. Droplet digital PCR detection of the erythropoietin transgene from horse plasma and urine for gene-doping control. Genes (Basel) 2019;10(3):243.
    doi: 10.3390/genes10030243google scholar: lookup
  19. Tozaki T, Ohnuma A, Kikuchi M. Microfluidic quantitative PCR detection of 12 transgenes from horse plasma for gene doping control. Genes (Basel) 2020;11(4):457.
    doi: 10.3390/genes11040457google scholar: lookup
  20. Tozaki T, Ohnuma A, Takasu M. Detection of non-targeted transgenes by whole-genome resequencing for gene-doping control. Gene Ther 2021;28(3-4):199-205.
    doi: 10.1038/s41434-020-00185-ygoogle scholar: lookup
  21. World Anti-Doping Agency. Laboratory Guidelines-Gene Doping Detection based on Polymerase Chain Reaction (PCR). Accessed January 18, 2023. https://www.wada-ama.org/en/resources/laboratory-guidelines-gene-doping-detection-based-polymerase-chain-reaction-pcr
  22. Association of Official Racing Chemists. AORC Guideline for the Minimum Criteria for Identification of Transgenes or Vectors by Polymerase Chain Reaction (PCR) Analysis. Restricted accessed January 18, 2023. https://www.aorc-online.org/members/
  23. Bray MS, Hagberg JM, Pérusse L. The human gene map for performance and health-related fitness phenotypes: the 2006-2007 update. Med Sci Sports Exerc 2009;41(1):35-73.
    doi: 10.1249/mss.0b013e3181844179google scholar: lookup
  24. Wilkin T, Baoutina A, Hamilton N. Equine performance genes and the future of doping in horseracing. Drug Test Anal 2017;9(9):1456-1471.
    doi: 10.1002/dta.2198google scholar: lookup
  25. Kalbfleisch TS, Rice ES, DePriest MS Jr. Improved reference genome for the domestic horse increases assembly contiguity and composition. Commun Biol 2018;1(1):197.
    doi: 10.1038/s42003-018-0199-zgoogle scholar: lookup
  26. Nurk S, Koren S, Rhie A. The complete sequence of a human genome. Science 2022;376(6588):44-53.
    doi: 10.1126/science.abj6987google scholar: lookup
  27. Wade CM, Giulotto E, Sigurdsson S. Genome sequence, comparative analysis, and population genetics of the domestic horse. Science 2009;326(5954):865-867.
    doi: 10.1126/science.1178158google scholar: lookup
  28. European Commission, Joint Research Centre. Guidance document on multiplex real-time PCR methods. Accessed January 18, 2023. https://data.europa.eu/doi/10.2760/243914
  29. Mahony JB, Petrich A, Smieja M. Molecular diagnosis of respiratory virus infections. Crit Rev Clin Lab Sci 2011;48(5-6):217-249.
    doi: 10.3109/10408363.2011.640976google scholar: lookup
  30. Parker J, Fowler N, Walmsley ML. Analytical sensitivity comparison between singleplex real-time PCR and a multiplex PCR platform for detecting respiratory viruses. PLoS ONE 2015;10(11):e0143164.
    doi: 10.1371/journal.pone.0143164google scholar: lookup
  31. Butler JM. Short tandem repeat typing technologies used in human identity testing. Biotechniques 2007;43(4):ii-v.
    doi: 10.2144/000112582google scholar: lookup
  32. Krüger J, Schleinitz D. Genetic fingerprinting using microsatellite markers in a multiplex PCR reaction: a compilation of methodological approaches from primer design to detection systems. Methods Mol Biol 2017;1492:1-15.
    doi: 10.1007/978-1-4939-6442-0_1google scholar: lookup
  33. International Organization for Standardization. ISO 20395:2019 Biotechnology-Requirements for Evaluating the Performance of Quantification Methods for Nucleic Acid Target Sequences-qPCR and dPCR. Accessed January 18, 2023. https://www.iso.org/standard/67893.html
  34. Bustin SA, Wittwer CT. MIQE: a step toward more robust and reproducible quantitative PCR. Clin Chem 2017;63(9):1537-1538.
    doi: 10.1373/clinchem.2016.268953google scholar: lookup
  35. Lewis C, Seashols-Williams SJ. Design and optimization of a 16S microbial qPCR multiplex for the presumptive identification of feces, saliva, vaginal and menstrual secretions. J Forensic Sci 2022;67(4):1660-1667.
    doi: 10.1111/1556-4029.15029google scholar: lookup
  36. Sánchez I, Dashti A, Köster PC. Development, optimisation and validation of a novel multiplex real-time PCR method for the simultaneous detection of Cryptosporidium spp., Giardia duodenalis and Dientamoeba fragilis. Pathogens 2022;11(11):1277.
    doi: 10.3390/pathogens11111277google scholar: lookup
  37. Sint D, Raso L, Traugott M. Advances in multiplex PCR: balancing primer efficiencies and improving detection success. Methods Ecol Evol 2012;3(5):898-905.
    doi: 10.1111/j.2041-210x.2012.00215.xgoogle scholar: lookup

Citations

This article has been cited 5 times.
  1. 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
  2. Wu D, Ding S, Liu N, Shi Y, Su P, Shi H, Shi Y, Han B, Cheng S, Ren X, Tian F, Chen P, Wu J, Su X, Li R. Codon changes challenge PCR-based gene doping detection. Gene Ther 2025 Dec;32(6):632-640.
    doi: 10.1038/s41434-025-00569-ypubmed: 41057515google scholar: lookup
  3. 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
  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.
    doi: 10.1038/s41434-024-00462-0pubmed: 38972888google scholar: lookup
  5. Wilkin T, Hamilton NA, Cawley AT, Bhat S, Baoutina A. PCR-Based Equine Gene Doping Test for the Australian Horseracing Industry. Int J Mol Sci 2024 Feb 22;25(5).
    doi: 10.3390/ijms25052570pubmed: 38473816google scholar: lookup
Subscribe to our newsletter
Join 99,001 horse owners like you who receive our email updates on horse health and nutrition.