PCR-Based Equine Gene Doping Test for the Australian Horseracing Industry
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: <i>EPO</i>, <i>FST</i>, <i>GH1</i>, <i>IGF1,</i> and <i>ILRN1</i>. T...
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| Format: | Article |
| Language: | English |
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MDPI AG
2024-02-01
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| Series: | International Journal of Molecular Sciences |
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| Online Access: | https://www.mdpi.com/1422-0067/25/5/2570 |
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| author | Tessa Wilkin Natasha A. Hamilton Adam T. Cawley Somanath Bhat Anna Baoutina |
| author_facet | Tessa Wilkin Natasha A. Hamilton Adam T. Cawley Somanath Bhat Anna Baoutina |
| author_sort | Tessa Wilkin |
| collection | DOAJ |
| description | 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: <i>EPO</i>, <i>FST</i>, <i>GH1</i>, <i>IGF1,</i> and <i>ILRN1</i>. 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. |
| first_indexed | 2024-04-25T00:28:46Z |
| format | Article |
| id | doaj.art-4d6069f11935434b9c98a516b188b534 |
| institution | Directory Open Access Journal |
| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-04-25T00:28:46Z |
| publishDate | 2024-02-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | International Journal of Molecular Sciences |
| spelling | doaj.art-4d6069f11935434b9c98a516b188b5342024-03-12T16:45:28ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672024-02-01255257010.3390/ijms25052570PCR-Based Equine Gene Doping Test for the Australian Horseracing IndustryTessa Wilkin0Natasha A. Hamilton1Adam T. Cawley2Somanath Bhat3Anna Baoutina4National Measurement Institute, Lindfield, NSW 2070, AustraliaFaculty of Veterinary Science, University of Sydney, Camperdown, NSW 2006, AustraliaAustralian Racing Forensic Laboratory, Racing NSW, Sydney, NSW 2000, AustraliaNational Measurement Institute, Lindfield, NSW 2070, AustraliaNational Measurement Institute, Lindfield, NSW 2070, AustraliaThe 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: <i>EPO</i>, <i>FST</i>, <i>GH1</i>, <i>IGF1,</i> and <i>ILRN1</i>. 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.https://www.mdpi.com/1422-0067/25/5/2570gene dopingreal-time PCRhorseracing |
| spellingShingle | Tessa Wilkin Natasha A. Hamilton Adam T. Cawley Somanath Bhat Anna Baoutina PCR-Based Equine Gene Doping Test for the Australian Horseracing Industry International Journal of Molecular Sciences gene doping real-time PCR horseracing |
| title | PCR-Based Equine Gene Doping Test for the Australian Horseracing Industry |
| title_full | PCR-Based Equine Gene Doping Test for the Australian Horseracing Industry |
| title_fullStr | PCR-Based Equine Gene Doping Test for the Australian Horseracing Industry |
| title_full_unstemmed | PCR-Based Equine Gene Doping Test for the Australian Horseracing Industry |
| title_short | PCR-Based Equine Gene Doping Test for the Australian Horseracing Industry |
| title_sort | pcr based equine gene doping test for the australian horseracing industry |
| topic | gene doping real-time PCR horseracing |
| url | https://www.mdpi.com/1422-0067/25/5/2570 |
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