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Detection using chamber digital PCR with a DNA extraction-free method for gene-doping control.

Risako Furukawa¹, Teruaki Tozaki¹, Koki Kawate¹

¹Genetic Analysis Department, Laboratory of Racing Chemistry, Utsunomiya, Tochigi, Japan.

Forensic Science International

|July 1, 2025

Summary

A new direct chamber digital PCR method detects gene doping in horse plasma without DNA purification. This rapid technique enhances equine sports integrity and forensic biotechnology applications.

Keywords:

Chamber digital PCR DNA extraction-free method Erythropoietin Gene doping Thoroughbred Transgene detection

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Area of Science:

Biotechnology
Forensic Science
Veterinary Medicine

Background:

Gene doping, using transgenes, threatens equine sports integrity and raises ethical concerns.
Current detection methods require time-consuming DNA extraction from plasma.
A simplified, rapid detection method is needed for gene doping surveillance.

Purpose of the Study:

To develop and validate a direct chamber digital PCR (cdPCR) method for transgene detection in equine plasma.
To eliminate the need for DNA purification, reducing processing time and labor.
To assess the utility of cdPCR for equine gene doping surveillance.

Main Methods:

Developed a direct chamber digital PCR (cdPCR) assay for transgene detection in equine plasma.
Used equine erythropoietin (EPO) transgene as a model, analyzing spiked plasma samples (10-1000 copies/µL).
Pre-treated samples with Lysis Buffer S Ver.2, using 0.55 µL plasma per 10 µL reaction.

Main Results:

The cdPCR method detected the EPO transgene in plasma without DNA purification.
Consistent detection down to 1000 copies/µL was achieved with high reproducibility and low background.
The assay successfully detected the EPO transgene in a horse post-plasmid administration.

Conclusions:

Direct cdPCR offers a practical and efficient tool for equine gene doping surveillance.
The simplified workflow and minimal sample requirement are advantageous for sports integrity and forensic applications.
This method significantly reduces sample handling and processing time compared to conventional PCR techniques.