Direct sequence confirmation of qPCR products for gene doping assay validation in horses

Maniego, Jillian; Pesko, Bogusia; Hincks, Pamela; Taylor, Polly; Stewart, Graham R.; Proudman, C. J.; Scarth, James; Ryder, Edward

doi:10.1002/dta.3219

Cited by 9 publications

(5 citation statements)

References 33 publications

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“…For example, Ryder’s group successfully detected gene doping in horses with the help of quantitative real-time PCR (qPCR) [ 68 ]. Two approaches, including the ligation of sequence-ready adapters to qPCR products and qPCR assays using tailed primers, were applied to provide direct analysis of the amplified qPCR products from five candidate genes by next-generation sequencing without adopting additional amplification techniques.…”

Section: Gene Doping Detectionmentioning

confidence: 99%

A Review of Recent Progress in Drug Doping and Gene Doping Control Analysis

Yan

et al. 2023

Molecules

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The illicit utilization of performance-enhancing substances, commonly referred to as doping, not only infringes upon the principles of fair competition within athletic pursuits but also poses significant health hazards to athletes. Doping control analysis has emerged as a conventional approach to ensuring equity and integrity in sports. Over the past few decades, extensive advancements have been made in doping control analysis methods, catering to the escalating need for qualitative and quantitative analysis of numerous banned substances exhibiting diverse chemical and biological characteristics. Progress in science, technology, and instrumentation has facilitated the proliferation of varied techniques for detecting doping. In this comprehensive review, we present a succinct overview of recent research developments within the last ten years pertaining to these doping detection methodologies. We undertake a comparative analysis, evaluating the merits and limitations of each technique, and offer insights into the prospective future advancements in doping detection methods. It is noteworthy that the continual design and synthesis of novel synthetic doping agents have compelled researchers to constantly refine and innovate doping detection methods in order to address the ever-expanding range of covertly employed doping agents. Overall, we remain in a passive position for doping detection and are always on the road to doping control.

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Section: Gene Doping Detectionmentioning

confidence: 99%

A Review of Recent Progress in Drug Doping and Gene Doping Control Analysis

Yan

et al. 2023

Molecules

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“…94 Combining PCR with NGS enables verification that the amplified PCR products correspond to the targeted transgene and has been validated using equine actinin alpha 3 (ACTN3), erythropoietin (EPO), hypoxia-inducible factor 1 subunit alpha (HIF1A), peroxisome proliferator-activated receptor delta (PPARD), and vascular endothelial growth factor A (VEGFA) transgenes spiked into equine plasma. 95 Other NGS-based methods have been developed to screen for up to 31 transgene or vectorspecific sequences simultaneously by combining PCR with massively parallel sequencing (MPS) using pooled primer sets. 96 For detection of gene editing, a non-targeted method was developed to identify transgenes as intron deletions using whole genome sequencing (WGS) and a structural variant detection software.…”

Section: In Vivo Raav Gene Therapy In Horsesmentioning

confidence: 99%

“…Digital droplet PCR (ddPCR) has been shown to detect very low copy numbers of transgenes spiked into equine plasma 93 particularly if a pre‐amplification step is used in a nested PCR method 94 . Combining PCR with NGS enables verification that the amplified PCR products correspond to the targeted transgene and has been validated using equine actinin alpha 3 (ACTN3), erythropoietin (EPO), hypoxia‐inducible factor 1 subunit alpha (HIF1A), peroxisome proliferator‐activated receptor delta (PPARD), and vascular endothelial growth factor A (VEGFA) transgenes spiked into equine plasma 95 . Other NGS‐based methods have been developed to screen for up to 31 transgene or vector‐specific sequences simultaneously by combining PCR with massively parallel sequencing (MPS) using pooled primer sets 96 …”

Section: Introductionmentioning

confidence: 99%

Administration and detection of gene therapy in horses: A systematic review

Haughan

Ortved

Robinson

2022

Drug Testing and Analysis

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Gene therapy uses genetic modification of cells to produce a therapeutic effect.Defective or missing genes can be repaired or replaced, or gene expression can be modified using a variety of technologies. Repair of defective genes can be achieved using specialized gene editing tools. Gene addition promotes gene expression by introducing synthetic copies of genes of interest (transgenes) into cells where they are transcribed and translated into therapeutic proteins. Protein production can also be modified using therapies that regulate gene expression.Gene therapy is currently prohibited in both human and equine athletes because of the potential to induce production of performance-enhancing proteins in the athlete's body, also referred to as "gene doping." Detection of gene doping is challenging and necessitates development of creative, novel analytical methods for doping control. Methods for detection of gene doping must be specific to and will vary depending on the type of gene therapy. The purpose of this paper is to present the results of a systematic review of gene editing, gene therapy, and detection of gene doping in horses.Based on the published literature, gene therapy has been administered to horses in a large number of experimental studies and a smaller number of clinical cases. Detection of gene therapy is possible using a combination of PCR and sequencing technologies. This summary can provide a basis for discussion of appropriate and inappropriate uses for gene therapy in horses by the veterinary community and guide expansion of methods to detect inappropriate uses by the regulatory community.

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“…Using NGS, transgenes can be detected as deletions of introns [ 27 , 28 , 29 ], enabling screening for nontargeted transgenes by comparing the detected intron deletions to annotated gene information (i.e., the intron location in reference sequences). Amplicon sequencing to detect targeted genes using NGS has also been developed for transgene detection [ 30 , 31 ].…”

Section: Introductionmentioning

confidence: 99%

Detection of Indiscriminate Genetic Manipulation in Thoroughbred Racehorses by Targeted Resequencing for Gene-Doping Control

Tozaki

Ohnuma

Nakamura

et al. 2022

Genes

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The creation of genetically modified horses is prohibited in horse racing as it falls under the banner of gene doping. In this study, we developed a test to detect gene editing based on amplicon sequencing using next-generation sequencing (NGS). We designed 1012 amplicons to target 52 genes (481 exons) and 147 single-nucleotide variants (SNVs). NGS analyses showed that 97.7% of the targeted exons were sequenced to sufficient coverage (depth > 50) for calling variants. The targets of artificial editing were defined as homozygous alternative (HomoALT) and compound heterozygous alternative (ALT1/ALT2) insertion/deletion (INDEL) mutations in this study. Four models of gene editing (three homoALT with 1-bp insertions, one REF/ALT with 77-bp deletion) were constructed by editing the myostatin gene in horse fibroblasts using CRISPR/Cas9. The edited cells and 101 samples from thoroughbred horses were screened using the developed test, which was capable of identifying the three homoALT cells containing 1-bp insertions. Furthermore, 147 SNVs were investigated for their utility in confirming biological parentage. Of these, 120 SNVs were amenable to consistent and accurate genotyping. Surrogate (nonbiological) dams were excluded by 9.8 SNVs on average, indicating that the 120 SNV could be used to detect foals that have been produced by somatic cloning or embryo transfer, two practices that are prohibited in thoroughbred racing and breeding. These results indicate that gene-editing tests that include variant calling and SNV genotyping are useful to identify genetically modified racehorses.

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Direct sequence confirmation of qPCR products for gene doping assay validation in horses

Cited by 9 publications

References 33 publications

A Review of Recent Progress in Drug Doping and Gene Doping Control Analysis

A Review of Recent Progress in Drug Doping and Gene Doping Control Analysis

Administration and detection of gene therapy in horses: A systematic review

Detection of Indiscriminate Genetic Manipulation in Thoroughbred Racehorses by Targeted Resequencing for Gene-Doping Control

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