A duplex qPCR assay for human erythropoietin (EPO) transgene to control gene doping in horses

Cheung, Hiu Wing; Wong, Kin‐Sing; Lin, Venus Y.C.; Wan, Terence S.M.; Ho, Emmie N.M.

doi:10.1002/dta.2907

Cited by 26 publications

(53 citation statements)

References 27 publications

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“…Gene doping in horseracing can be divided into two categories: (1) administration of gene-doping substances to postnatal animals and (2) the generation of genetically modified racehorses. Although detection methods for the former have been developed 11 – 16 , no methods are available for detecting genetic modifications. For this purpose, whole-genome sequencing 17 , which requires a detailed understanding of variations of the Thoroughbred genome, may be useful.…”

Section: Introductionmentioning

confidence: 99%

Rare and common variant discovery by whole-genome sequencing of 101 Thoroughbred racehorses

Tozaki

Ohnuma

Kikuchi

et al. 2021

Sci Rep

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The Thoroughbred breed was formed by crossing Oriental horse breeds and British native horses and is currently used in horseracing worldwide. In this study, we constructed a single-nucleotide variant (SNV) database using data from 101 Thoroughbred racehorses. Whole genome sequencing (WGS) revealed 11,570,312 and 602,756 SNVs in autosomal (1–31) and X chromosomes, respectively, yielding a total of 12,173,068 SNVs. About 6.9% of identified SNVs were rare variants observed only in one allele in 101 horses. The number of SNVs detected in individual horses ranged from 4.8 to 5.3 million. Individual horses had a maximum of 25,554 rare variants; several of these were functional variants, such as non-synonymous substitutions, start-gained, start-lost, stop-gained, and stop-lost variants. Therefore, these rare variants may affect differences in traits and phenotypes among individuals. When observing the distribution of rare variants among horses, one breeding stallion had a smaller number of rare variants compared to other horses, suggesting that the frequency of rare variants in the Japanese Thoroughbred population increases through breeding. In addition, our variant database may provide useful basic information for industrial applications, such as the detection of genetically modified racehorses in gene-doping control and pedigree-registration of racehorses using SNVs as markers.

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Section: Introductionmentioning

confidence: 99%

Rare and common variant discovery by whole-genome sequencing of 101 Thoroughbred racehorses

Tozaki

Ohnuma

Kikuchi

et al. 2021

Sci Rep

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“…Bone tissue defects are cured by the morphogenesis, cell differentiation and regeneration and repair of the tissue. 9 The repair of bone tissue is different from other tissue repairs, not only in the potential of bone tissue to differentiate itself. What is important is that tissues other than bone can produce osteogenesis under the action of osteoinductive factors.…”

Section: Discussionmentioning

confidence: 99%

Physical Exercise in the Promotion of Gene Therapy Auxiliary Effect

Shen

Liang

2021

Rev Bras Med Esporte

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Introduction: In recent years, genetic engineering has made outstanding contributions to sports, and it has played a huge role in promoting the development of sports-related fields. Objective: We analyze the tissue source of bone growth and healing by studying the role of bone morphogenetic protein and transforming growth factors in fracture injuries caused by sports. Methods: We established a human fracture model to express the shape and content of bone morphogenetic protein and transforming growth factor during fracture healing. Results: In the fracture healing stage caused by different sports, the expression levels of the two genes are different. Bone morphogenetic protein has a high content in the osteogenesis stage of the membrane, while transforming growth factor is high in the cartilage ossification stage. Conclusion: Gene therapy for fractures caused by physical exercise has certain advantages. Osteoblasts and chondrocytes are involved in the synthesis of transforming growth factors. Level of evidence II; Therapeutic studies - investigation of treatment results.

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“…Several transgene detection methods have been developed and reported 5–9 . While some use next‐generation sequencing technology, 10,11 most transgene detection methods are based on polymerase chain reaction (PCR), real‐time PCR and digital PCR.…”

Section: Introductionmentioning

confidence: 99%

Low‐copy transgene detection using nested digital polymerase chain reaction for gene‐doping control

Tozaki

Ohnuma

Hamilton

et al. 2021

Drug Testing and Analysis

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Gene doping is prohibited for fair competition in human and horse sports. One style of gene doping is the administration of an exogeneous gene, called a transgene, to postnatal humans and horses. Although many transgene detection methods based on quantitative polymerase chain reaction (PCR), including real-time PCR and digital PCR, have been recently developed, it remains difficult to reliably detect low-copy transgenes. In this study, we developed and validated a nested digital PCR method to specifically detect low-copy transgenes. The nested digital PCR consists of (1) preamplification using conventional PCR and (2) droplet digital PCR detection using a hydrolysis probe. Using 5, 10, 20, 60 and 120 transgene copies as template, 496.0, 1089.7, 1820.7, 4313.3 and 7840.0 copies per microlitre, respectively, were detected using our nested digital PCR. Although high concentrations of phenol, proteinase K, ethanol, EDTA, heparin and genomic DNA all inhibited preamplification, their effects on the digital PCR detection were limited. Once preamplification was successful, even substitution of bases within the primers and probes had minimal effects on transgene detection. The nested digital PCR developed in this study successfully detected lowcopy transgenes and can be used to perform a qualitative test, indicating its usefulness in the prevention of false positives and false negatives in gene-doping detection.

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A duplex qPCR assay for human erythropoietin (EPO) transgene to control gene doping in horses

Cited by 26 publications

References 27 publications

Rare and common variant discovery by whole-genome sequencing of 101 Thoroughbred racehorses

Rare and common variant discovery by whole-genome sequencing of 101 Thoroughbred racehorses

Physical Exercise in the Promotion of Gene Therapy Auxiliary Effect

Low‐copy transgene detection using nested digital polymerase chain reaction for gene‐doping control

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