scholarly journals Detection of Multiple Transgene Fragments in a Mouse Model of Gene Doping Based on Plasmid Vector Using TaqMan-qPCR Assay

2020 ◽  
Author(s):  
Takeito Sugasawa ◽  
Kai Aoki ◽  
Koki Yanazawa ◽  
Ryo Hagino ◽  
Shinsuke Tamai ◽  
...  
Keyword(s):  
Whole Blood ◽  
Abdominal Cavity ◽  
Plasmid Vector ◽  
Firefly Luciferase ◽  
Qpcr Assay ◽  
Gold Standard Method ◽  
Gene Doping ◽  
Taqman Qpcr Assay ◽  
Taqman Qpcr

The World Anti-Doping Agency has prohibited gene doping in the context of progress in gene therapy. There is a risk that the artificial regulation of genes using plasmids could be applied for gene doping. However, no gold standard method to detect this has been established. Here, we aimed to develop a method to detect multiple transgene fragments as proof of gene doping. First, gene delivery model mice as a mimic of gene doping were created by injecting firefly luciferase plasmid with polyethylenimine (PEI) into the abdominal cavity. The results confirmed successful establishment of the model, with sufficient luminescence upon in vivo imaging. Next, multiple transgene fragments in the model were detected in plasma cell-free (cf)DNA, blood-cell-fraction DNA, and stool DNA using the TaqMan-qPCR assay, with the highest levels in plasma cfDNA. Using just a single drop of whole blood from the model, we also attempted long-term detection. The results showed that multiple transgene fragments were detected until 11 days. These findings indicate that the combination of plasma cfDNA or just one drop of whole blood with TaqMan-qPCR assay is feasible to detect plasmid-PEI-based gene doping. Our findings could accelerate the development of methods for detecting gene doping in humans.

scholarly journals Detection of Multiple Transgene Fragments in a Mouse Model of Gene Doping Based on Plasmid Vector Using TaqMan-qPCR Assay

Genes ◽  
2020 ◽  
Vol 11 (7) ◽  
pp. 750
Author(s):  
Takehito Sugasawa ◽  
Kai Aoki ◽  
Kouki Yanazawa ◽  
Kazuhiro Takekoshi
Keyword(s):  
Whole Blood ◽  
Abdominal Cavity ◽  
Plasmid Vector ◽  
Firefly Luciferase ◽  
Qpcr Assay ◽  
Gold Standard Method ◽  
Gene Doping ◽  
Taqman Qpcr Assay ◽  
Taqman Qpcr

The World Anti-Doping Agency has prohibited gene doping in the context of progress in gene therapy. There is a risk that the augmentation of genes using plasmids could be applied for gene doping. However, no gold standard method to detect this has been established. Here, we aimed to develop a method to detect multiple transgene fragments as proof of gene doping. Firstly, gene delivery model mice as a mimic of gene doping were created by injecting firefly luciferase plasmid with polyethylenimine (PEI) into the abdominal cavity. The results confirmed successful establishment of the model, with sufficient luminescence upon in vivo imaging. Next, multiple transgene fragments in the model were detected in plasma cell-free (cf)DNA, blood-cell-fraction DNA, and stool DNA using the TaqMan- quantitative real-time PCR(qPCR) assay, with the highest levels in plasma cfDNA. Using just a single drop of whole blood from the model, we also attempted long-term detection. The results showed that multiple transgene fragments were detected until 11 days. These findings indicate that the combination of plasma cfDNA or just one drop of whole blood with TaqMan-qPCR assay is feasible to detect plasmid-PEI-based gene doping. Our findings could accelerate the development of methods for detecting gene doping in humans.

scholarly journals The detection of trans gene fragments of hEPO in gene doping model mice by Taqman qPCR assay

2019 ◽  
Author(s):  
Kai Aoki ◽  
Takehito Sugasawa ◽  
Kouki Yanazawa ◽  
Koichi Watanabe ◽  
Tohru Takemasa ◽  
...  
Keyword(s):  
Whole Blood ◽  
Plasmid Vector ◽  
Sybr Green ◽  
Gene Doping ◽  
Human Erythropoietin ◽  
World Anti Doping Agency ◽  
Total Dna ◽  
Stool Dna ◽  
Tail Tip ◽  
Taqman Qpcr

BACKGROUND. With the rapid progress of genetic engineering and gene therapy methods, the World Anti-Doping Agency has raised concerns regarding gene doping, which is prohibited in sports. However, there is no standard method available for detecting transgenes delivered by injection of naked plasmids. Here, we developed a detection method for detecting transgenes delivered by injection of naked plasmids in a mouse model that mimics gene doping. METHODS. Whole blood from the tail tip and one piece of stool were used as pre-samples of injection. Next, a plasmid vector containing the human erythropoietin (hEPO) gene was injected into mice through intravenous (IV), intraperitoneal (IP), or local muscular (IM) injection. At 1, 2, 3, 6, 12, 24, and 48 h after injection, approximately 50 μL whole blood was collected from the tail tip. One piece of stool was collected at 6, 12, 24, and 48 h. From each sample, total DNA was extracted and transgene fragments were analyzed by Taqman quantitative PCR (qPCR) and SYBR green qPCR. RESULTS. In whole blood DNA samples evaluated by Taqman qPCR, the transgene fragments were detected at all time points in the IP sample and at 1, 2, 3, 6, and 12 h in the IV and IM samples. In the stool-DNA samples, the transgene fragments were detected at 6, 12, 24, and 48 h in the IV and IM samples by Taqman qPCR. In the analysis by SYBR green qPCR, the transgene fragments were detected at some time point in both specimens; however, many non-specific amplicons were detected. CONCLUSIONS. These results indicate that transgene fragments evaluated after each injection method of naked plasmids were detected in whole-blood and stool DNA samples. These findings may facilitate the development of methods for detecting gene doping.

scholarly journals The detection of trans gene fragments of hEPO in gene doping model mice by Taqman qPCR assay

PeerJ ◽  
2020 ◽  
Vol 8 ◽  
pp. e8595 ◽  
Author(s):  
Kai Aoki ◽  
Takehito Sugasawa ◽  
Kouki Yanazawa ◽  
Koichi Watanabe ◽  
Tohru Takemasa ◽  
...  
Keyword(s):  
Whole Blood ◽  
Plasmid Vector ◽  
Sybr Green ◽  
Gene Doping ◽  
Human Erythropoietin ◽  
World Anti Doping Agency ◽  
Total Dna ◽  
Stool Dna ◽  
Tail Tip ◽  
Taqman Qpcr

Background With the rapid progress of genetic engineering and gene therapy methods, the World Anti-Doping Agency has raised concerns regarding gene doping, which is prohibited in sports. However, there is no standard method available for detecting transgenes delivered by injection of naked plasmids. Here, we developed a detection method for detecting transgenes delivered by injection of naked plasmids in a mouse model that mimics gene doping. Methods Whole blood from the tail tip and one piece of stool were used as pre-samples of injection. Next, a plasmid vector containing the human erythropoietin (hEPO) gene was injected into mice through intravenous (IV), intraperitoneal (IP), or local muscular (IM) injection. At 1, 2, 3, 6, 12, 24, and 48 h after injection, approximately 50 µL whole blood was collected from the tail tip. One piece of stool was collected at 6, 12, 24, and 48 h. From each sample, total DNA was extracted and transgene fragments were analyzed by Taqman quantitative PCR (qPCR) and SYBR green qPCR. Results In whole blood DNA samples evaluated by Taqman qPCR, the transgene fragments were detected at all time points in the IP sample and at 1, 2, 3, 6, and 12 h in the IV and IM samples. In the stool-DNA samples, the transgene fragments were detected at 6, 12, 24, and 48 h in the IV and IM samples by Taqman qPCR. In the analysis by SYBR green qPCR, the transgene fragments were detected at some time point in both specimens; however, many non-specific amplicons were detected. Conclusions These results indicate that transgene fragments evaluated after each injection method of naked plasmids were detected in whole-blood and stool DNA samples. These findings may facilitate the development of methods for detecting gene doping.

scholarly journals The detection of trans gene fragments of hEPO in gene doping model mice by Taqman qPCR assay

2019 ◽  
Author(s):  
Kai Aoki ◽  
Takehito Sugasawa ◽  
Kouki Yanazawa ◽  
Koichi Watanabe ◽  
Tohru Takemasa ◽  
...  
Keyword(s):  
Whole Blood ◽  
Plasmid Vector ◽  
Sybr Green ◽  
Gene Doping ◽  
Human Erythropoietin ◽  
World Anti Doping Agency ◽  
Total Dna ◽  
Stool Dna ◽  
Tail Tip ◽  
Taqman Qpcr

BACKGROUND. With the rapid progress of genetic engineering and gene therapy methods, the World Anti-Doping Agency has raised concerns regarding gene doping, which is prohibited in sports. However, there is no standard method available for detecting transgenes delivered by injection of naked plasmids. Here, we developed a detection method for detecting transgenes delivered by injection of naked plasmids in a mouse model that mimics gene doping. METHODS. Whole blood from the tail tip and one piece of stool were used as pre-samples of injection. Next, a plasmid vector containing the human erythropoietin (hEPO) gene was injected into mice through intravenous (IV), intraperitoneal (IP), or local muscular (IM) injection. At 1, 2, 3, 6, 12, 24, and 48 h after injection, approximately 50 μL whole blood was collected from the tail tip. One piece of stool was collected at 6, 12, 24, and 48 h. From each sample, total DNA was extracted and transgene fragments were analyzed by Taqman quantitative PCR (qPCR) and SYBR green qPCR. RESULTS. In whole blood DNA samples evaluated by Taqman qPCR, the transgene fragments were detected at all time points in the IP sample and at 1, 2, 3, 6, and 12 h in the IV and IM samples. In the stool-DNA samples, the transgene fragments were detected at 6, 12, 24, and 48 h in the IV and IM samples by Taqman qPCR. In the analysis by SYBR green qPCR, the transgene fragments were detected at some time point in both specimens; however, many non-specific amplicons were detected. CONCLUSIONS. These results indicate that transgene fragments evaluated after each injection method of naked plasmids were detected in whole-blood and stool DNA samples. These findings may facilitate the development of methods for detecting gene doping.

scholarly journals Dynamics of Specific cfDNA Fragments in the Plasma of Full Marathon Participants

Genes ◽  
2021 ◽  
Vol 12 (5) ◽  
pp. 676
Author(s):  
Takehito Sugasawa ◽  
Shin-ichiro Fujita ◽  
Tomoaki Kuji ◽  
Noriyo Ishibashi ◽  
Kenshirou Tamai ◽  
...  
Keyword(s):  
Exercise Physiology ◽  
High Sensitivity ◽  
Genomic Region ◽  
Qpcr Assay ◽  
Strongly Correlated ◽  
Healthy Humans ◽  
Blood Cell And Plasma ◽  
Next Generation Sequencing Ngs ◽  
Taqman Qpcr Assay ◽  
Taqman Qpcr

Plasma cell-free DNA (cfDNA) is frequently analyzed using liquid biopsy to investigate cancer markers. We hypothesized that this concept might be applicable in exercise physiology. Here, we aimed to identify specific cfDNA (spcfDNA) sequences in the plasma of healthy humans using next-generation sequencing (NGS) and clearly define the dynamics regarding spcfDNA-fragment levels upon extreme exercises, such as running a full marathon. NGS analysis was performed using cfDNA of pooled plasma collected from healthy participants. We confirmed that the TaqMan-qPCR assay had high sensitivity and found that the spcfDNA sequence abundance was 16,600-fold higher than that in a normal genomic region. We then used the TaqMan-qPCR assay to investigate the dynamics of spcfDNA-fragment levels upon running a full marathon. The spcfDNA fragment levels were significantly increased post-marathon. Furthermore, spcfDNA fragment levels were strongly correlated with white blood cell and plasma myoglobin concentrations. These results suggest the spcfDNA fragments identified in this study were highly sensitive as markers of extreme physical stress. The findings of this study may provide new insights into exercise physiology and genome biology in humans.

scholarly journals A multiplex TaqMan qPCR assay for sensitive and rapid detection of phytoplasmas infecting Rubus species

PLoS ONE ◽  
2017 ◽  
Vol 12 (5) ◽  
pp. e0177808 ◽  
Author(s):  
Holger Linck ◽  
Erika Krüger ◽  
Annette Reineke
Keyword(s):  
Rapid Detection ◽  
Qpcr Assay ◽  
Rubus Species ◽  
Taqman Qpcr Assay ◽  
Taqman Qpcr

Development and evaluation of a direct TaqMan qPCR assay for the rapid detection of diverse carnivore amdoparvoviruses

2019 ◽  
Vol 48 ◽  
pp. 101448
Author(s):  
Yan-Hong Wu ◽  
Tao Wei ◽  
Xiu-Ting Zhang ◽  
Yong-Qiang Zhao ◽  
Jian-Ke Wang ◽  
...  
Keyword(s):  
Rapid Detection ◽  
Qpcr Assay ◽  
Taqman Qpcr Assay ◽  
Taqman Qpcr
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