Low‐copy transgene detection using nested digital PCR for gene doping control

2021 ◽  
Author(s):  
Teruaki Tozaki ◽  
Aoi Ohnuma ◽  
Natasha A. Hamilton ◽  
Mio Kikuchi ◽  
Taichiro Ishige ◽  
...  
Keyword(s):  
Digital Pcr ◽  
Doping Control ◽  
Gene Doping ◽  
Transgene Detection

Robustness of Digital PCR and Real-Time PCR in Transgene Detection for Gene-Doping Control

2021 ◽  
Author(s):  
Teruaki Tozaki ◽  
Aoi Ohnuma ◽  
Shinichi Iwai ◽  
Mio Kikuchi ◽  
Taichiro Ishige ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Digital Pcr ◽  
Doping Control ◽  
Gene Doping ◽  
Transgene Detection

Simulated validation of intron‐less transgene detection using DELLY for gene‐doping control in horse sports

2021 ◽  
Author(s):  
T. Tozaki ◽  
A. Ohnuma ◽  
M. Kikuchi ◽  
T. Ishige ◽  
H. Kakoi ◽  
...  
Keyword(s):  
Doping Control ◽  
Gene Doping ◽  
Transgene Detection

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

Genes ◽  
2019 ◽  
Vol 10 (3) ◽  
pp. 243 ◽  
Author(s):  
Teruaki Tozaki ◽  
Aoi Ohnuma ◽  
Masaki Takasu ◽  
Mio Kikuchi ◽  
Hironaga Kakoi ◽  
...  
Keyword(s):  
Magnetic Beads ◽  
Genetic Manipulation ◽  
Digital Pcr ◽  
Absolute Quantification ◽  
Droplet Digital Pcr ◽  
Taqman Probe ◽  
Gene Doping ◽  
Athletic Ability ◽  
Horse Plasma ◽  
Primer Sets

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.

scholarly journals Detection of Transgenes in Gene Delivery Model Mice by Adenoviral Vector Using ddPCR

2019 ◽  
Author(s):  
Takehito Sugasawa ◽  
Kai Aoki ◽  
Koichi Watanabe ◽  
Koki Yanazawa ◽  
Tohru Natsume ◽  
...  
Keyword(s):  
Blood Cell ◽  
Quantitative Pcr ◽  
Whole Blood ◽  
Digital Pcr ◽  
Cell Fraction ◽  
Detection Methods ◽  
Gene Doping ◽  
World Anti Doping Agency ◽  
Injection Methods ◽  
Tail Tip

With the rapid progress of genetic engineering and gene therapy, World Anti-Doping Agency has alerted to gene doping and prohibited its use in sports. However, there is no standard method available yet for detection of transgenes delivered by recombinant adenoviral (rAdV) vectors. Here we aimed to develop a detection method for transgenes delivered by rAdV vectors in a mouse model that mimics gene doping. rAdV vectors containing mCherry gene was delivered in mice through intravenous injection or local muscular injection. After five days, stool and whole blood samples were collected, and total DNA was extracted. As additional experiments, whole blood was also collected from mouse tail tip until 15 days from injection of the rAdv vector. Transgene fragments from different DNA samples were analyzed using semi-quantitative PCR (sqPCR), quantitative PCR (qPCR), and droplet digital PCR (ddPCR). In the results, transgene fragments could directly be detected from blood cell fraction-DNA, plasma-cell free DNA and stool-DNA by qPCR and ddPCR, depending on specimen type and injection methods. We observed that a combination of blood cell fraction-DNA and ddPCR was more sensitive than other combinations used in this model. These results could accelerate the development of detection methods for gene doping.

scholarly journals Detection of Transgenes in Gene Delivery Model Mice by Adenoviral Vector Using ddPCR

Genes ◽  
2019 ◽  
Vol 10 (6) ◽  
pp. 436 ◽  
Author(s):  
Takehito Sugasawa ◽  
Kai Aoki ◽  
Koichi Watanabe ◽  
Koki Yanazawa ◽  
Tohru Natsume ◽  
...  
Keyword(s):  
Blood Cell ◽  
Quantitative Pcr ◽  
Whole Blood ◽  
Digital Pcr ◽  
Cell Fraction ◽  
Detection Methods ◽  
Gene Doping ◽  
World Anti Doping Agency ◽  
Injection Methods ◽  
Tail Tip

With the rapid progress of genetic engineering and gene therapy, the World Anti-Doping Agency has been alerted to gene doping and prohibited its use in sports. However, there is no standard method available yet for the detection of transgenes delivered by recombinant adenoviral (rAdV) vectors. Here, we aim to develop a detection method for transgenes delivered by rAdV vectors in a mouse model that mimics gene doping. These rAdV vectors containing the mCherry gene was delivered in mice through intravenous injection or local muscular injection. After five days, stool and whole blood samples were collected, and total DNA was extracted. As additional experiments, whole blood was also collected from the mouse tail tip until 15 days from injection of the rAdv vector. Transgene fragments from different DNA samples were analyzed using semi-quantitative PCR (sqPCR), quantitative PCR (qPCR), and droplet digital PCR (ddPCR). In the results, transgene fragments could be directly detected from blood cell fraction DNA, plasma cell-free DNA, and stool DNA by qPCR and ddPCR, depending on specimen type and injection methods. We observed that a combination of blood cell fraction DNA and ddPCR was more sensitive than other combinations used in this model. These results could accelerate the development of detection methods for gene doping.

scholarly journals Digital PCR detection of plasmid DNA administered to the skeletal muscle of a microminipig: a model case study for gene doping detection

2018 ◽  
Vol 11 (1) ◽  
Author(s):  
Teruaki Tozaki ◽  
Shiori Gamo ◽  
Masaki Takasu ◽  
Mio Kikuchi ◽  
Hironaga Kakoi ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Plasmid Dna ◽  
Digital Pcr ◽  
Pcr Detection ◽  
Model Case ◽  
Gene Doping

scholarly journals Gene Doping Control in Horseracing Industries

2021 ◽  
Vol 49 (1) ◽  
pp. 19-29
Author(s):  
Teruaki TOZAKI ◽  
Aoi OHNUMA ◽  
Mio KIKUCHI ◽  
Taichiro ISHIGE ◽  
Kei-ichi HIROTA ◽  
...  
Keyword(s):  
Doping Control ◽  
Gene Doping

scholarly journals Whole-genome resequencing using genomic DNA extracted from horsehair roots for gene-doping control in horse sports

2020 ◽  
Vol 31 (4) ◽  
pp. 75-83
Author(s):  
Teruaki TOZAKI ◽  
Aoi OHNUMA ◽  
Mio KIKUCHI ◽  
Taichiro ISHIGE ◽  
Hironaga KAKOI ◽  
...  
Keyword(s):  
Genomic Dna ◽  
Doping Control ◽  
Whole Genome ◽  
Genome Resequencing ◽  
Gene Doping ◽  
Whole Genome Resequencing

scholarly journals Microfluidic Quantitative PCR Detection of 12 Transgenes from Horse Plasma for Gene Doping Control

Genes ◽  
2020 ◽  
Vol 11 (4) ◽  
pp. 457 ◽  
Author(s):  
Teruaki Tozaki ◽  
Aoi Ohnuma ◽  
Mio Kikuchi ◽  
Taichiro Ishige ◽  
Hironaga Kakoi ◽  
...  
Keyword(s):  
Quantitative Pcr ◽  
Limit Of Detection ◽  
Doping Control ◽  
Taqman Probe ◽  
Specific Detection ◽  
Gene Doping ◽  
Athletic Ability ◽  
Horse Plasma ◽  
Fluidic Circuits ◽  
Primer Sets

Gene doping, an activity which abuses and misuses gene therapy, is a major concern in sports and horseracing industries. Effective methods capable of detecting and monitoring gene doping are urgently needed. Although several PCR-based methods that detect transgenes have been developed, many of them focus only on a single transgene. However, numerous genes associated with athletic ability may be potential gene-doping material. Here, we developed a detection method that targets multiple transgenes. We targeted 12 genes that may be associated with athletic performance and designed two TaqMan probe/primer sets for each one. A panel of 24 assays was prepared and detected via a microfluidic quantitative PCR (MFQPCR) system using integrated fluidic circuits (IFCs). The limit of detection of the panel was 6.25 copy/μL. Amplification-specificity was validated using several concentrations of reference materials and animal genomic DNA, leading to specific detection. In addition, target-specific detection was successfully achieved in a horse administered 20 mg of the EPO transgene via MFQPCR. Therefore, MFQPCR may be considered a suitable method for multiple-target detection in gene-doping control. To our knowledge, this is the first application of microfluidic qPCR (MFQPCR) for gene-doping control in horseracing.

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