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Droplet Digital PCR Detection of the Erythropoietin Transgene from Horse Plasma and Urine for Gene-Doping Control

Indiscriminate genetic manipulation to improve athletic ability is a major threat to human sports and the horseracing industry, in which methods involving gene-doping, such as transgenesis, should be prohibited to ensure fairness. Therefore, development of methods to detect indiscriminate genetic ma...

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Autores principales: Tozaki, Teruaki, Ohnuma, Aoi, Takasu, Masaki, Kikuchi, Mio, Kakoi, Hironaga, Hirota, Kei-ichi, Kusano, Kanichi, Nagata, Shun-ichi
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6471249/
https://www.ncbi.nlm.nih.gov/pubmed/30901981
http://dx.doi.org/10.3390/genes10030243
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author Tozaki, Teruaki
Ohnuma, Aoi
Takasu, Masaki
Kikuchi, Mio
Kakoi, Hironaga
Hirota, Kei-ichi
Kusano, Kanichi
Nagata, Shun-ichi
author_facet Tozaki, Teruaki
Ohnuma, Aoi
Takasu, Masaki
Kikuchi, Mio
Kakoi, Hironaga
Hirota, Kei-ichi
Kusano, Kanichi
Nagata, Shun-ichi
author_sort Tozaki, Teruaki
collection PubMed
description Indiscriminate genetic manipulation to improve athletic ability is a major threat to human sports and the horseracing industry, in which methods involving gene-doping, such as transgenesis, should be prohibited to ensure fairness. Therefore, development of methods to detect indiscriminate genetic manipulation are urgently needed. Here, we developed a highly sensitive method to detect horse erythropoietin (EPO) transgenes using droplet digital PCR (ddPCR). We designed two TaqMan probe/primer sets, and the EPO transgene was cloned into a plasmid for use as a model. We extracted the spiked EPO transgene from horse plasma and urine via magnetic beads, followed by ddPCR amplification for absolute quantification and transgene detection. The results indicated high recovery rates (at least ~60% and ~40% in plasma and urine, respectively), suggesting successful detection of the spiked transgene at concentrations of >130 and 200 copies/mL of plasma and urine, respectively. Additionally, successful detection was achieved following intramuscular injection of 20 mg of the EPO transgene. This represents the first study demonstrating a method for detecting the EPO transgene in horse plasma and urine, with our results demonstrating its efficacy for promoting the control of gene-doping in the horseracing industry.
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spelling pubmed-64712492019-04-27 Droplet Digital PCR Detection of the Erythropoietin Transgene from Horse Plasma and Urine for Gene-Doping Control Tozaki, Teruaki Ohnuma, Aoi Takasu, Masaki Kikuchi, Mio Kakoi, Hironaga Hirota, Kei-ichi Kusano, Kanichi Nagata, Shun-ichi Genes (Basel) Article Indiscriminate genetic manipulation to improve athletic ability is a major threat to human sports and the horseracing industry, in which methods involving gene-doping, such as transgenesis, should be prohibited to ensure fairness. Therefore, development of methods to detect indiscriminate genetic manipulation are urgently needed. Here, we developed a highly sensitive method to detect horse erythropoietin (EPO) transgenes using droplet digital PCR (ddPCR). We designed two TaqMan probe/primer sets, and the EPO transgene was cloned into a plasmid for use as a model. We extracted the spiked EPO transgene from horse plasma and urine via magnetic beads, followed by ddPCR amplification for absolute quantification and transgene detection. The results indicated high recovery rates (at least ~60% and ~40% in plasma and urine, respectively), suggesting successful detection of the spiked transgene at concentrations of >130 and 200 copies/mL of plasma and urine, respectively. Additionally, successful detection was achieved following intramuscular injection of 20 mg of the EPO transgene. This represents the first study demonstrating a method for detecting the EPO transgene in horse plasma and urine, with our results demonstrating its efficacy for promoting the control of gene-doping in the horseracing industry. MDPI 2019-03-21 /pmc/articles/PMC6471249/ /pubmed/30901981 http://dx.doi.org/10.3390/genes10030243 Text en © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Tozaki, Teruaki
Ohnuma, Aoi
Takasu, Masaki
Kikuchi, Mio
Kakoi, Hironaga
Hirota, Kei-ichi
Kusano, Kanichi
Nagata, Shun-ichi
Droplet Digital PCR Detection of the Erythropoietin Transgene from Horse Plasma and Urine for Gene-Doping Control
title Droplet Digital PCR Detection of the Erythropoietin Transgene from Horse Plasma and Urine for Gene-Doping Control
title_full Droplet Digital PCR Detection of the Erythropoietin Transgene from Horse Plasma and Urine for Gene-Doping Control
title_fullStr Droplet Digital PCR Detection of the Erythropoietin Transgene from Horse Plasma and Urine for Gene-Doping Control
title_full_unstemmed Droplet Digital PCR Detection of the Erythropoietin Transgene from Horse Plasma and Urine for Gene-Doping Control
title_short Droplet Digital PCR Detection of the Erythropoietin Transgene from Horse Plasma and Urine for Gene-Doping Control
title_sort droplet digital pcr detection of the erythropoietin transgene from horse plasma and urine for gene-doping control
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6471249/
https://www.ncbi.nlm.nih.gov/pubmed/30901981
http://dx.doi.org/10.3390/genes10030243
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