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

doi:10.3390/genes10030243

Cited by 39 publications

(79 citation statements)

References 17 publications

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“…The plasma showed signals indicating high concentrations of the EPO transgene at 15 min, 3 h, 6 h, 12 h and 24 h following its administration (Figure 4; yellow signal) and signals indicating low concentrations 2 d after administration (Figure 4; purple signal). These concentration trends were similar to those seen in one of our previous studies [16], in which ddPCR was used to detect the EPO transgene. All the signals from 15 min to 2 d were over the LOD (6.25 copy/µL).…”

Section: Detection Of the Epo Transgene After It Was Administered To supporting

confidence: 87%

“…Thus, confirmation experiments should follow such screenings. Our previous study [16] developed and evaluated a gene-doping detection method using ddPCR, which is able to quantify gene-doping materials. Therefore, a combination of MFQPCR for screening and ddPCR for confirmation may be more suitable for the detection of gene doping under certain circumstances.…”

Section: Discussionmentioning

confidence: 99%

“…Sequences of the cloned transgenes were confirmed via Sanger sequencing. In addition, the EPO transgene administered to horses in this study was prepared according to a method in our previous study [16]. Briefly, a 579-bp horse EPO containing ORF (192 amino acids) and a termination codon was cloned into a plasmid vector pBApo-CMV (r-Amp + , CMV IE promoter, HSV TK poly A; Takara Bio).…”

Section: Preparation Of Cloned Horse Transgenes As Reference Materialsmentioning

confidence: 99%

“…The 12 transgenes dissolved in Milli-Q water were quantified using a NanoDrop One C , Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific) and droplet digital PCR (ddPCR; Bio-Rad, Hercules, CA, USA) using a TaqMan probe. For ddPCR, we followed a protocol used in our previous study [16]. Finally, values quantified by ddPCR were used for concentrations of reference material (RM) in the current study.…”

Section: Quantitation Of Transgenes Cloned Into Plasmid Vectormentioning

confidence: 99%

“…The number of animals used was kept to a minimum in this case study in order to comply with animal ethics and welfare guidelines. Administration of the EPO transgene to a horse has been previously performed by us [16]. Briefly, a thoroughbred horse (male, 11 years old, 465 kg) at the ERI, JRA was intramuscularly injected with 20 mg of the horse EPO transgene, and blood samples were collected as follows: before administration, 15 min, 3 h, 6 h, 12 h, 24 h, 2 d, 3 d, 4 d, 5 d, 6 d, 7 d, 14 d, 21 d and 28 d after administration.…”

Section: Administration Of Epo Transgenementioning

confidence: 99%

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

Tozaki

Ohnuma

Kikuchi

et al. 2020

Genes

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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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Section: Detection Of the Epo Transgene After It Was Administered To supporting

confidence: 87%

Section: Discussionmentioning

confidence: 99%

Section: Preparation Of Cloned Horse Transgenes As Reference Materialsmentioning

confidence: 99%

Section: Quantitation Of Transgenes Cloned Into Plasmid Vectormentioning

confidence: 99%

Section: Administration Of Epo Transgenementioning

confidence: 99%

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

Tozaki

Ohnuma

Kikuchi

et al. 2020

Genes

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Synthesis of a biocompatible fluorinated multiblock copolymer surfactant for water‐in‐fluorocarbon droplets and its application in droplet digital polymerase chain reaction

Feng,

Li,

Zhu

et al. 2024

J of Applied Polymer Sci

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Droplet digital polymerase chain reaction (ddPCR) technology has attracted considerable attention in recent years. A multiblock surfactant based on perfluompolyethers (PFPE) has been widely used in droplet‐based microfluidics and is known to provide high droplet stability and biocompatibility. We developed a multiblock copolymer surfactant synthesized by adjusting the proportions of the intermediates to enhance the stabilities of droplets at high temperatures. The surfactants were synthesized via amidation reactions that coupled the PFPE and diamine polyethylene glycol (PEG), and the introduction of triethylamine, as a catalyst, made the reaction more efficient. The as‐synthesized surfactants were used to generate water‐in‐ fluorocarbon (W/F) droplets with a microfluidic device and a ddPCR device was used to evaluate the performance of the droplets. When the molar ratio (PFPE‐PEG:PFPE‐PEG‐PFPE) was 2:1 and the concentration reached 2 wt%, the droplets showed good thermal stability. Surfactant showed the best performance, lowering the interfacial tension to 2.5 mN/m, when it's critical micelle concentration (CMC) exceeded 50 mg/L. Furthermore, the use of an alkaline solution (pH = 9–10) as the washing buffer, improved the biocompatible of the surfactant. When applied to the detection of λDNA, the limit of detection was 0.52 copies/μL. The as‐synthesized surfactants showed promise in generating biocompatible and thermally stable droplets and providing a new surfactant option for future ddPCR technology.

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Detection of intra‐articular gene therapy in horses using quantitative real time PCR in synovial fluid and plasma

Haughan

Jiang

Stefanovski

et al. 2020

Drug Testing and Analysis

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Gene therapy promotes the expression of missing or defective genes and can be curative following administration of a single dose. Gene therapy is prohibited in equine athletes by regulatory bodies due to the high potential for abuse and novel analytical methods are needed for detection. The goal of this study was to detect the administration of an experimental gene therapy: a recombinant adeno‐associated viral vector (rAAV) carrying a transgene for the anti‐inflammatory cytokine IL‐10 (rAAV‐IL10). Twelve horses were randomly assigned to receive an intra‐articular injection of rAAV‐IL10 or phosphate buffered saline (vehicle) into a middle carpal joint. Plasma and synovial fluid were collected on days 0, 1, 2, 4, 7, 14, 28, 56, and 84. Primer pairs were designed to detect two unique regions of rAAV. Using quantitative real time PCR, both sets of primers detected rAAV for 14–28 days in joints and up to 4 days in plasma, in all six treated horses. In synovial fluid, rAAV was detected on day 56 in 4/6 horses by both primer sets, and on day 84 in 1/6 horses by one primer set. In plasma, rAAV was detected for 7 days in 5/6 horses, 14 days in 2/6 horses, and 28 days in 1/6 horses by one primer set, and was detected for up to 14 days in 1/6 horses by the other primer set. This study is the first to validate that quantitative real time PCR can be used to systemically detect the local administration of a gene therapy product to horses.

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

Cited by 39 publications

References 17 publications

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

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

Synthesis of a biocompatible fluorinated multiblock copolymer surfactant for water‐in‐fluorocarbon droplets and its application in droplet digital polymerase chain reaction

Detection of intra‐articular gene therapy in horses using quantitative real time PCR in synovial fluid and plasma

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