A method for detecting gene doping in horse sports without DNA extraction

Area of Science:

• Veterinary Science
• Molecular Biology
• Biotechnology

Background:

• Gene doping, involving transgenes, is a prohibited practice in equine sports.
• Current detection methods like quantitative polymerase chain reaction (qPCR) require DNA extraction from plasma, which is time-consuming and sample-intensive.
• There is a need for more efficient and sensitive methods to detect gene doping in horses.

Purpose of the Study:

• To develop and validate novel DNA-extraction-free methods for detecting the equine erythropoietin (EPO) transgene.
• To assess the sensitivity and applicability of direct and nested droplet digital PCR (ddPCR) for gene doping detection in equine plasma.

Main Methods:

• Development of direct ddPCR using pretreated plasma to detect spiked EPO transgenes at 10 copies/μL.
• Development of nested ddPCR involving pre-amplification, PCR product purification, and PCR for detecting spiked EPO transgenes at 1 copy/μL.
• Application and validation of both methods using plasma samples from horses administered the EPO transgene.

Main Results:

• Both direct and nested ddPCR methods successfully detected the equine EPO transgene after intramuscular injection.
• Nested ddPCR demonstrated higher sensitivity, detecting the transgene at 1 copy/μL.
• The methods require minimal sample volumes (2.2-5.0 μL) due to the absence of a DNA extraction step.

Conclusions:

• Direct and nested ddPCR offer reliable, sensitive, and efficient alternatives to conventional gene doping detection methods.
• Combining direct ddPCR for screening and nested ddPCR for confirmation can enhance accuracy and prevent false positives.
• These novel methods facilitate the detection of gene doping in equine sports using small sample volumes.