EPO transgene detection in dried blood spots for antidoping application

Area of Science:

• Biotechnology
• Sports Science
• Molecular Biology

Background:

• Gene expression modification is a rapidly advancing field with implications for both disease treatment and performance enhancement.
• The surveillance of gene expression modification for athletic performance enhancement, particularly targeting erythropoietin (EPO), is increasingly necessary.
• Existing methods for EPO gene doping detection focus on identifying transgenes different in size from endogenous sequences.

Purpose of the Study:

• To validate an easily applicable method for detecting EPO gene doping using dried blood spots (DBS).
• To evaluate the sensitivity and reliability of EPO transgene detection in DBS samples.

Main Methods:

• Evaluated EPO transgene detection in 20-μl DBS samples spiked with an EPO-carrying plasmid.
• Compared three DBS types: Nucleic-Card™, Whatman® 903, and volumetric 20-μl VAMS™.
• Utilized real-time polymerase chain reaction (PCR) with custom and commercial Taqman assays for detection and validation.

Main Results:

• Successfully detected 1000 copies of EPO transgene from 20-μl DBS using all three tested materials, with VAMS™ showing slightly better sensitivity.
• No loss of detection sensitivity was observed after 1-month of DBS storage at room temperature.
• The validated method allows for efficient detection and reanalysis of EPO gene doping in DBS.

Conclusions:

• The developed method provides a sensitive and reliable approach for EPO gene doping detection in DBS.
• This technique is suitable for application in anti-doping controls, offering a practical solution for sample collection and analysis.
• The ability to use DBS facilitates wider sample collection and supports robust anti-doping efforts in sports.