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Development of Detection Method Using Dried Blood Spot with Next-Generation Sequencing and LabDroid for Gene Doping

Kiyoshi Maehara1, Atsushi Hirokawa2, Hinata Watanabe2

  • 1Doctoral Program in Medical Sciences, Graduate School of Comprehensive Human Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba 305-8572, Japan.

International Journal of Molecular Sciences
|July 12, 2025
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Summary

Researchers developed a new gene doping detection method using dried blood spots (DBS) and next-generation sequencing (NGS). This innovative approach successfully identified gene doping in a mouse model, paving the way for improved anti-doping strategies.

Keywords:
LabDroidMaholoNGSdried blood spoterythropoietingene doping

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Area of Science:

  • Sports Science
  • Biotechnology
  • Genetics

Background:

  • Gene therapy advancements raise concerns about potential misuse in sports doping.
  • Current gene doping detection methods have limitations and require enhancement.
  • Progress in blood storage, next-generation sequencing (NGS), and robotics offers new possibilities.

Purpose of the Study:

  • To develop an improved method for detecting gene doping.
  • To utilize dried blood spot (DBS) technology for sample collection.
  • To integrate NGS and LabDroid automation for enhanced detection capabilities.

Main Methods:

  • Established a gene doping mouse model using recombinant adeno-associated virus carrying the human erythropoietin (hEPO) gene.
  • Collected whole blood and prepared dried blood spots (DBS).
  • Employed the LabDroid "Maholo" for DNA extraction from DBS and library preparation for NGS, followed by bioinformatic analysis.

Main Results:

  • Successfully identified specific DNA fragments indicative of hEPO gene doping in the treated mice.
  • The developed method demonstrated clear differentiation between the gene-doped model and control mice.
  • Confirmed the feasibility of using DBS, Maholo, and NGS for gene doping detection.

Conclusions:

  • This study presents the first biological model of hEPO gene doping utilizing Maholo, NGS, and DBS.
  • The integrated method shows significant promise for advancing the development of reliable gene doping tests.
  • This approach could enhance anti-doping efforts in sports by providing a more robust detection system.