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Multiplex detection of seven transgenes for human gene doping analysis
Nana Naumann1, Carolin Do2, Caren Vollmert3
1Center for Preventive Doping Research, Institute of Biochemistry, German Sport University Cologne, Cologne, Germany. n.naumann@biochem.dshs-koeln.de.
Scientific Reports
|June 20, 2025
Summary
A new multiplex assay combining PCR and MALDI-TOF MS can detect seven human transgenes used for gene doping. This method offers high sensitivity and specificity for enhanced athletic performance detection in sports drug testing.
Area of Science:
- Biotechnology
- Molecular Biology
- Sports Science
Background:
- Gene doping involves manipulating genes for athletic advantage, posing a challenge to anti-doping efforts.
- Current detection methods may lack the multiplexing capability required for comprehensive screening.
Purpose of the Study:
- To develop and validate a multiplex assay for detecting multiple gene doping transgenes simultaneously.
- To assess the sensitivity, specificity, and applicability of the assay in human sports drug testing.
Main Methods:
- Development of a multiplex Polymerase Chain Reaction (PCR) assay combined with Matrix-Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS).
- The panel targets seven human transgenes (EPO, FST, GH1, IGF1, MSTN, VEGFA, VEGFD) via exon-exon-junction detection.
- A reference material (RM) was also designed and tested for assay validation.
Main Results:
- The multiplex panel demonstrated high sensitivity with a limit of detection (LOD95) of 1,500 copies/mL for the panel and 500 copies/mL for the RM.
- Specificity was confirmed through testing of equine plasma and 111 native human doping control samples.
- The assay successfully detected rAAV-delivered human transgenic EPO in equine plasma.
Conclusions:
- The developed multiplex assay is a sensitive and specific tool for detecting multiple gene doping agents.
- This assay has practical applicability for routine human sports drug testing programs.
- The assay represents a significant advancement in gene doping detection technology.
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