PCR-Based Equine Gene Doping Test for the Australian Horseracing Industry.
Abstract: The term 'gene doping' is used to describe the use of any unauthorized gene therapy techniques. We developed a test for five likely candidate genes for equine gene doping: , , , and . The test is based on real-time polymerase chain reaction (PCR) and includes separate screening and confirmation assays that detect different unique targets in each transgene. For doping material, we used nonviral (plasmid) and viral (recombinant adeno-associated virus) vectors carrying complementary DNA for the targeted genes; the vectors were accurately quantified by digital PCR. To reduce non-specific amplification from genomic DNA observed in some assays, a restriction digest step was introduced in the PCR protocol prior to cycling to cut the amplifiable targets within the endogenous genes. We made the screening stage of the test simpler and faster by multiplexing PCR assays for four transgenes (EPO, FST, IGF1, and ILRN1), while the GH1 assay is performed in simplex. Both stages of the test reliably detect at least 20 copies of each transgene in a background of genomic DNA equivalent to what is extracted from two milliliters of equine blood. The test protocol was documented and tested with equine blood samples provided by an official doping control authority. The developed tests will form the basis for screening official horseracing samples in Australia.
Publication Date: 2024-02-22 PubMed ID: 38473816DOI: 10.3390/ijms25052570Google Scholar: Lookup
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Summary
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This research article discusses the creation of a reliable genetic testing method for possible gene doping in racehorses, helping create a level playing field in the horse racing industry.
Understanding Gene Doping
- The practice of gene doping involves using unapproved gene therapy techniques, often with the aim of enhancing an animal or individual’s performance.
- Sporting authorities seek to prevent this form of cheating to maintain the integrity and health of the sport.
Development of a New Test Method
- The researchers focused on five probable genes (EPO, FST, IGF1, ILRN1, and GH1) susceptible to doping in equines.
- To identify any modifications to these genes, they built a test that utilizes real-time polymerase chain reaction (PCR) – a technique that amplifies and simultaneously monitors DNA replication in real time.
- The test involves unique screening and confirmation assays that discern different distinctive targets within each altered gene.
Enhancing Test Accuracy
- They applied nonviral (plasmid) and viral (recombinant adeno-associated virus) vectors carrying complementary DNA of the targeted genes as the basis for doping material.
- These vectors were precisely quantified using digital PCR, enhancing the reliability of the test results.
- A reduction in non-specific amplification from genomic DNA was achieved by introducing a restriction digest step into the PCR protocol before cycling.
Simplifying the Test Procedure
- To streamline the process, the team multiplicatively ran PCR assays for four transgenes: EPO, FST, IGF1, and ILRN1. The GH1 assay, however, was run individually.
- These tests detected at least 20 copies of each transgene within a genomic DNA background equivalent to that extracted from two milliliters of equine blood, asserting the tests’ sensitivity.
Bridging the Gap in Horse Racing
- The test procedure was documented and assessed using equine blood samples supplied by a recognized doping control authority.
- These developed tests will now become a standard part of screening for the detection of gene doping in the official horseracing samples in Australia.
Cite This Article
APA
Wilkin T, Hamilton NA, Cawley AT, Bhat S, Baoutina A.
(2024).
PCR-Based Equine Gene Doping Test for the Australian Horseracing Industry.
Int J Mol Sci, 25(5), 2570.
https://doi.org/10.3390/ijms25052570 Publication
Researcher Affiliations
- National Measurement Institute, Lindfield, NSW 2070, Australia.
- Faculty of Veterinary Science, University of Sydney, Camperdown, NSW 2006, Australia.
- Faculty of Veterinary Science, University of Sydney, Camperdown, NSW 2006, Australia.
- Equine Genetics Research Centre, Racing Australia, Sydney, NSW 2000, Australia.
- Australian Racing Forensic Laboratory, Racing NSW, Sydney, NSW 2000, Australia.
- Racing Analytical Services Limited, Flemington, VIC 3031, Australia.
- National Measurement Institute, Lindfield, NSW 2070, Australia.
- National Measurement Institute, Lindfield, NSW 2070, Australia.
MeSH Terms
- Animals
- Horses / genetics
- Erythropoietin / genetics
- Australia
- Plasmids
- DNA / genetics
- Real-Time Polymerase Chain Reaction
Grant Funding
- The detection of gene doping in Thoroughbred racehorses / Racing Australia
Citations
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