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Codon changes challenge PCR-based gene doping detection

Gene Therapy (2025)Cite this article

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Abstract

Genetic/genomic manipulation techniques (gene transfer/delivery, gene editing, etc.) have become more and more mature, and the illegal use as gene doping in sports has drawn attentions. World Anti-Doping Agency (WADA) strictly prohibits gene doping, and has issued guideline on quantitative real-time PCR (qPCR) detections. However, the technical feature of qPCR makes it difficult to detect new doping targets, and codon changes on targets may also affect detection efficiency. Here, we prepare standard materials for genomic and transgenic versions of human EPO (hEPO) gene, and design qPCR primers to check the consequences of codon changes on gene doping detection. We confirm that carefully designed qPCR assays could indeed capture transgene signal, but codon changes on the transgene could severely undermine detection efficiency. We have also mimicked real world gene doping scenario by mixing genomic and transgenic versions of hEPO, and qPCR could detect wild-type but not codon-changed transgenes. As a method validation for such a challenge, we also use Sanger sequencing to confirm that sequencing could easily capture gene doping even for codon-changed transgenes. Our study confirms that codon changes will challenge qPCR-based gene doping detection, and calls for un-biased detection tools based on high-throughput sequencing in the future.

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Fig. 1: Design of qPCR assays for gene doping detection.
Fig. 2: Codon changes undermine gene doping detection by qPCR.
Fig. 3: Codon changes enable detection escape in mimicked gene doping scenario.
Fig. 4: Sequencing could capture gene doping even with codon changes.
Fig. 5: Summary of the main findings.

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All the data are provided in the manuscript.

References

  1. Doudna JA. The promise and challenge of therapeutic genome editing. Nature. 2020;578:229–36.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Dunbar CE, High KA, Joung JK, Kohn DB, Ozawa K, Sadelain M. Gene therapy comes of age. Science. 2018;359:175.

    Article  CAS  Google Scholar 

  3. Sun W, Wang H. Recent advances of genome editing and related technologies in China. Gene Ther. 2020;27:312–20.

    Article  CAS  PubMed  Google Scholar 

  4. Wong C. UK first to approve CRISPR treatment for diseases: what you need to know. Nature. 2023;623:676–7.

    Article  CAS  PubMed  Google Scholar 

  5. Reardon S. It’s a vote for hope’: first gene therapy for muscular dystrophy nears approval, but will it work?. Nature. 2023;618:451–3.

    Article  CAS  Google Scholar 

  6. Lv J, Wang H, Cheng X, Chen Y, Wang D, Zhang L, et al. AAV1-hOTOF gene therapy for autosomal recessive deafness 9: a single-arm trial. Lancet. 2024;403:2317–25.

    Article  CAS  PubMed  Google Scholar 

  7. Musunuru K, Grandinette SA, Wang X, Hudson TR, Briseno K, Berry AM, et al. Patient-specific in vivo gene editing to treat a rare genetic disease. N Engl J Med. 2025;392:2235–43.

    Article  CAS  PubMed  Google Scholar 

  8. Filipp F. Is science killing sport? Gene therapy and its possible abuse in doping. Embo Rep. 2007;8:433–5.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Friedmann T, Rabin O, Frankel MS. Gene doping and sport. Science. 2010;327:647–8.

    Article  CAS  PubMed  Google Scholar 

  10. Pincock S. Gene doping. Lancet. 2005;366:S18–S9.

    Article  PubMed  Google Scholar 

  11. Li RH, Su PP, Shi Y, Shi H, Ding SQ, Su XB, et al. Gene doping detection in the era of genomics. Drug Test Anal. 2024;16:1468–78.

    Article  CAS  PubMed  Google Scholar 

  12. Lopez S, Meirelles J, Rayol V, Poralla G, Woldmar N, Fadel B, et al. Gene doping and genomic science in sports: where are we? Bioanalysis. 2020;12:801–11.

    Article  CAS  PubMed  Google Scholar 

  13. Gineviciene V, Utkus A, Pranckeviciene E, Semenova EA, Hall ECR, Ahmetov II. Perspectives in sports genomics. Biomedicines. 2022;10:298.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Baoutina A, Bhat S, Li DK, Emslie KR. Towards a robust test to detect gene doping for anabolic enhancement in human athletes. Drug Test Anal. 2023;15:314–23.

    Article  CAS  PubMed  Google Scholar 

  15. Yanazawa K, Sugasawa T, Aoki K, Nakano T, Kawakami Y, Takekoshi K. Development of a gene doping detection method to detect overexpressed human follistatin using an adenovirus vector in mice. PeerJ. 2021;9.e12285.

    Article  PubMed  PubMed Central  Google Scholar 

  16. Kaiser J. How safe is a popular gene therapy vector? Science. 2020;367:131.

    Article  CAS  PubMed  Google Scholar 

  17. Smalley E. FDA warns public of dangers of DIY gene therapy. Nat Biotechnol. 2018;36:119–20.

    Article  CAS  PubMed  Google Scholar 

  18. WADA. The Prohibited List. https://www.wada-ama.org/en/prohibited-list. 2025.

  19. Naumann N, Paßreiter A, Thomas A, Krug O, Walpurgis K, Thevis M. Analysis of potential gene doping preparations for transgenic DNA in the context of sports drug testing programs. Int J Mol Sci. 2023;24:15835.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Puchalska M, Witkowska-Piłaszewicz O. Gene doping in horse racing and equine sports: current landscape and future perspectives. Equine Vet J. 2025;57:312–24.

    Article  PubMed  Google Scholar 

  21. Tozaki T, Hamilton NA. Control of gene doping in human and horse sports. Gene Ther. 2022;29:107–12.

    Article  CAS  PubMed  Google Scholar 

  22. 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;92:16322–8.

    Article  PubMed  Google Scholar 

  23. Zheng B, Yan J, Li T, Zhao Y, Xu Z, Rao R, et al. Hydrophilic/hydrophobic modified microchip for detecting multiple gene doping candidates using CRISPR-Cas12a and RPA. Biosens Bioelectron. 2024;263:116631.

    Article  CAS  PubMed  Google Scholar 

  24. Paßreiter A, Naumann N, Thomas A, Grogna N, Delahaut P, Thevis M. Detection of sgRNA via SHERLOCK as potential CRISPR related gene doping control strategy. Anal Chem. 2024;96:7452–9.

    Article  PubMed  Google Scholar 

  25. Sugasawa T, Aoki K, Yanazawa K, Takekoshi K. Detection of multiple transgene fragments in a mouse model of gene doping based on plasmid vector using TaqMan-qPCR assay. Genes. 2020;11:750.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Neuberger EWI, Perez I, Le Guiner C, Moser D, Ehlert T, Allais M, et al. Establishment of two quantitative nested qPCR assays targeting the human EPO transgene. Gene Ther. 2016;23:330–9.

    Article  CAS  PubMed  Google Scholar 

  27. Tozaki T, Ohnuma A, Iwai S, Kikuchi M, Ishige T, Kakoi H, et al. Robustness of digital PCR and real-time PCR in transgene detection for gene-doping control. Anal Chem. 2021;93:7133–9.

    Article  CAS  PubMed  Google Scholar 

  28. Tozaki T, Ohnuma A, Kikuchi M, Ishige T, Kakoi H, Hirota KI, et al. Robustness of digital PCR and real-time PCR against inhibitors in transgene detection for gene doping control in equestrian sports. Drug Test Anal. 2021;13:1768–75.

    Article  CAS  PubMed  Google Scholar 

  29. Moser DA, Braga L, Raso A, Zacchigna S, Giacca M, Simon P. Transgene detection by digital droplet PCR. PLoS ONE. 2014;9:e111781.

    Article  PubMed  PubMed Central  Google Scholar 

  30. Ohnuma A, Tozaki T, Kikuchi M, Ishige T, Kakoi H, Hirota K-i, et al. Multiplex detection of transgenes using πcode technology for gene doping control. Anal Chem. 2023;95:10149–54.

    Article  CAS  PubMed  Google Scholar 

  31. Salamin O, Kuuranne T, Saugy M, Leuenberger N. Loop-mediated isothermal amplification (LAMP) as an alternative to PCR: a rapid on-site detection of gene doping. Drug Test Anal. 2017;9:1731–7.

    Article  CAS  PubMed  Google Scholar 

  32. WADA. Laboratory Guidelines-Gene Doping Detection based on Polymerase Chain Reaction (PCR). https://www.wada-ama.org/en/resources/laboratory-guidelines-gene-doping-detection-based-polymerase-chain-reaction-pcr. 2021.

  33. Naumann N, Do C, Vollmert C, Krajina M, Thomas A, Cheung HW, et al. Multiplex detection of seven transgenes for human gene doping analysis. Sci Rep. 2025;15:20219.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Wong KS, Cheung HW, Szeto CWL, Tsang CYN, Wan TSM, Ho ENM. A multiplex qPCR assay for transgenes detection: a novel approach for gene doping control in horseracing using conventional laboratory setup. Drug Test Anal. 2023;15:879–88.

    Article  CAS  PubMed  Google Scholar 

  35. Baoutina A, Bhat S, Zheng M, Partis L, Dobeson M, Alexander IE, et al. Synthetic certified DNA reference material for analysis of human erythropoietin transgene and transcript in gene doping and gene therapy. Gene Ther. 2016;23:708–17.

    Article  CAS  PubMed  Google Scholar 

  36. Baoutina A, Coldham T, Bains GS, Emslie KR. Gene doping detection: evaluation of approach for direct detection of gene transfer using erythropoietin as a model system. Gene Ther. 2010;17:1022–32.

    Article  CAS  PubMed  Google Scholar 

  37. Ostrander EA, Huson HJ, Ostrander GK. Genetics of athletic performance. Annu Rev Genomics Hum Genet. 2009;10:407–29.

    Article  CAS  PubMed  Google Scholar 

  38. Ren X, Shi Y, Xiao B, Su X, Shi H, He G, et al. Gene doping detection from the perspective of 3D genome. Drug Test Anal. 2025;17:1475–89.

  39. Chin JX, Chung BKS, Lee DY. Codon Optimization OnLine (COOL): a web-based multi-objective optimization platform for synthetic gene design. Bioinformatics. 2014;30:2210–2.

    Article  CAS  PubMed  Google Scholar 

  40. Hernandez-Alias X, Benisty H, Radusky LG, Serrano L, Schaefer MH. Using protein-per-mRNA differences among human tissues in codon optimization. Genome Biol. 2023;24:34.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Thevis M, Kuuranne T, Geyer H. Annual banned-substance review 17th edition—analytical approaches in human sports drug testing 2023/2024. Drug Test Anal. 2025;17:1417–42.

    Article  CAS  PubMed  Google Scholar 

  42. Su X, Shi Y, Li R, Lu ZN, Zou X, Wu JX, et al. Application of qPCR assays based on haloacids transporter gene dehp2 for discrimination of Burkholderia and Paraburkholderia. BMC Microbiol. 2019;19:36.

    Article  PubMed  PubMed Central  Google Scholar 

  43. Su X, Shi Y, Zou X, Lu Z-N, Xie G, Yang JYH, et al. Single-cell RNA-Seq analysis reveals dynamic trajectories during mouse liver development. BMC Genomics. 2017;18:946.

    Article  PubMed  PubMed Central  Google Scholar 

  44. Pfaffl MW. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res. 2001;29:e45.

  45. van der Gronde T, de Hon O, Haisma HJ, Pieters T. Gene doping: an overview and current implications for athletes. Br J Sports Med. 2013;47:670–8.

    Article  PubMed  Google Scholar 

  46. Wells DJ. Gene doping: the hype and the reality. Br J Pharmacol. 2008;154:623–31.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Thompson H. Performance enhancement: superhuman athletes. Nature. 2012;487:287–9.

    Article  CAS  PubMed  Google Scholar 

  48. Bara S, Barczuk P, Gałajda E, Antonik J, Barczak I, Barć J, et al. A review of the possibilities of gene doping in sports focused on the advantages and disadvantages. J Educ Health Sport. 2023;42:35–45.

    Article  Google Scholar 

  49. Baoutina A. A brief history of the development of a gene doping test. Bioanalysis. 2020;12:723–7.

    Article  CAS  PubMed  Google Scholar 

  50. Cantelmo RA, da Silva AP, Mendes-Junior CT, Dorta DJ. Gene doping: present and future. Eur J Sport Sci. 2020;20:1093–101.

    Article  PubMed  Google Scholar 

  51. Okano M, Ikekita A, Sato M, Inoue T, Kageyama S, Akiyama K, et al. Doping control analyses during the Tokyo 2020 Olympic and Paralympic Games. Drug Test Anal. 2022;14:1836–52.

    Article  CAS  PubMed  Google Scholar 

  52. Mauro VP, Chappell SA. A critical analysis of codon optimization in human therapeutics. Trends Mol Med. 2014;20:604–13.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Sugasawa T, Nakano T, Fujita S-i, Matsumoto Y, Ishihara G, Aoki K, et al. Proof of gene doping in a mouse model with a human erythropoietin gene transferred using an adenoviral vector. Genes. 2021;12:1249.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Beiter T, Zimmermann M, Fragasso A, Hudemann J, Niess AM, Bitzer M, et al. Direct and long-term detection of gene doping in conventional blood samples. Gene Ther. 2011;18:225–31.

    Article  CAS  PubMed  Google Scholar 

  55. Ni W, Le Guiner C, Gernoux G, Penaud-Budloo M, Moullier P, Snyder RO. Longevity of rAAV vector and plasmid DNA in blood after intramuscular injection in nonhuman primates: implications for gene doping. Gene Ther. 2011;18:709–18.

    Article  CAS  PubMed  Google Scholar 

  56. Lu Y, Yan J, Ou G, Fu L. A review of recent progress in drug doping and gene doping control analysis. Molecules. 2023;28:5483.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. de Boer EN, van der Wouden PE, Johansson LF, van Diemen CC, Haisma HJ. A next-generation sequencing method for gene doping detection that distinguishes low levels of plasmid DNA against a background of genomic DNA. Gene Ther. 2019;26:338–46.

    Article  PubMed  PubMed Central  Google Scholar 

  58. Tozaki T, Ohnuma A, Nakamura K, Hano K, Takasu M, Takahashi Y, et al. Detection of indiscriminate genetic manipulation in thoroughbred racehorses by targeted resequencing for gene-doping control. Genes. 2022;13:1589.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Tozaki T, Ohnuma A, Kikuchi M, Ishige T, Kakoi H, Hirota KI, et al. Whole-genome resequencing using genomic DNA extracted from horsehair roots for gene-doping control in horse sports. J Equine Sci. 2020;31:75–83.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Tozaki T, Ohnuma A, Takasu M, Nakamura K, Kikuchi M, Ishige T, et al. Detection of non-targeted transgenes by whole-genome resequencing for gene-doping control. Gene Ther. 2021;28:199–205.

    Article  CAS  PubMed  Google Scholar 

  61. Tozaki T, Ohnuma A, Kikuchi M, Ishige T, Kakoi H, Hirota K-i, et al. Rare and common variant discovery by whole-genome sequencing of 101 Thoroughbred racehorses. Sci Rep. 2021;11:16057.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Sutehall S, Malinsky F, Voss S, Chester N, Xu X, Pitsiladis Y. Practical steps to develop a transcriptomic test for blood doping. Transl Exerc Biomed. 2024;1:105–10.

    Article  Google Scholar 

  63. Logsdon GA, Vollger MR, Eichler EE. Long-read human genome sequencing and its applications. Nat Rev Genet. 2020;21:597–614.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. Maniego J, Giles O, Hincks P, Stewart G, Proudman C, Ryder E. Long-read sequencing assays designed to detect potential gene editing events in the myostatin gene revealed distinct haplotype signatures in the Thoroughbred horse population. Anim Genet. 2023;54:470–82.

    Article  CAS  PubMed  Google Scholar 

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Funding

This work is supported in part by Natural Science Foundation of Shanghai (25ZR1401192), Shanghai Gaofeng & Gaoyuan Project for University Academic Program Development (Shanghai University of Sport-2023), SJTU Interdisciplinary Program (YG2023QNB06), and Fundamental Research Funds for the Central Universities, Key Laboratory of Systems Biomedicine (Ministry of Education) Grant (KLSB2024QN-04).

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Author notes

  1. These authors contributed equally: Die Wu, Shengqian Ding, Nian Liu, Yi Shi.

Authors and Affiliations

  1. Shanghai Anti-doping Laboratory, Shanghai University of Sport, Shanghai, PR China

    Die Wu, Shengqian Ding, Nian Liu, Yi Shi, Hui Shi, Xinyuan Ren, Peijie Chen & Xianbin Su

  2. Key Laboratory of Systems Biomedicine (Ministry of Education), Shanghai Center for Systems Biomedicine, Shanghai Jiao Tong University, Shanghai, PR China

    Shengqian Ding, Nian Liu & Xianbin Su

  3. Hongqiao International Institute of Medicine, Tongren Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, PR China

    Shengqian Ding, Nian Liu, Bo Han, Sheng Cheng & Jiaoxiang Wu

  4. Bio-X Institutes, Key Laboratory for the Genetics of Developmental and Neuropsychiatric Disorders, Shanghai Jiao Tong University, Shanghai, PR China

    Yi Shi

  5. Innovative Program of Medicine, Shanghai Jiao Tong University School of Medicine, Shanghai, PR China

    Peipei Su

  6. Department of Rheumatology and Immunology, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, PR China

    Hui Shi

  7. School of Exercise and Health, Shanghai University of Sport, Shanghai, PR China

    Yue Shi

  8. Welsh School of Architecture, Cardiff University, Cardiff, UK

    Futong Tian

  9. Shanghai Center of Agri-Products Quality and Safety, Shanghai, PR China

    Ruihong Li

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  1. Die Wu
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Contributions

Conceptualization, original hypothesis, design of the study and methodology, XS; investigation, DW, SD, NL, YS1, PS, HS, YS2, BH, SC, XR, and FT; writing – original draft, RL; writing – review & editing, all authors; supervision, PC, JW, XS, and RL.

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Correspondence to Peijie Chen, Jiaoxiang Wu, Xianbin Su or Ruihong Li.

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Ethical approval was not needed for this paper as only synthesized DNA standard materials were used, without involvement of human or animal specimens/experiments.

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Wu, D., Ding, S., Liu, N. et al. Codon changes challenge PCR-based gene doping detection. Gene Ther (2025). https://doi.org/10.1038/s41434-025-00569-y

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