PCR-Based Equine Gene Doping Test for the Australian Horseracing Industry

Area of Science:

• Equine Sports Science
• Molecular Biology
• Anti-Doping Technology

Background:

• Gene doping, the illicit use of gene therapy, poses a significant threat to equine sports integrity.
• Identifying specific transgenes used for doping is crucial for effective detection.

Purpose of the Study:

• To develop and validate a sensitive real-time PCR-based test for detecting five candidate genes associated with equine gene doping.
• To establish a reliable screening and confirmation method for anti-doping laboratories.

Main Methods:

• Development of real-time PCR assays targeting five equine gene doping candidates: EPO, FST, GH1, IGF1, and ILRN1.
• Utilized both screening and confirmation assays with distinct targets for each transgene.
• Incorporated a restriction digest step to minimize non-specific amplification from genomic DNA.
• Employed multiplexing for faster screening of EPO, FST, IGF1, and ILRN1, with GH1 tested separately.
• Validated the test using viral and nonviral vectors containing target gene sequences and equine blood samples.

Main Results:

• The developed test reliably detects as few as 20 copies of each transgene in a background of genomic DNA equivalent to 2 mL of equine blood.
• The screening assay was optimized for speed and simplicity through multiplexing.
• Both screening and confirmation assays demonstrated high specificity and sensitivity.

Conclusions:

• The developed real-time PCR test provides a robust tool for detecting gene doping in racehorses.
• This assay is suitable for implementation in official anti-doping control programs, starting with Australian horseracing.
• The test contributes to maintaining fair competition and the welfare of equine athletes.