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

Haughan, Joanne; Jiang, Zibin; Stefanovski, Darko; Moss, Kaitlyn L.; Ortved, Kyla F.; Robinson, Mary Ann

doi:10.1002/dta.2785

Cited by 23 publications

(42 citation statements)

References 33 publications

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“…Various methods have been developed to detect transgenes using quantitative polymerase chain reaction (qPCR), loop‐mediated isothermal amplification and next‐generation sequencing (de Boer et al, 2019; Haughan et al, 2020; Maniego et al, 2021; Salamin et al, 2017; Tozaki et al, 2019, 2020). Recently, WADA published a laboratory guideline on PCR‐based transgene detection (WADA, 2021).…”

Section: Introductionmentioning

confidence: 99%

Identification of processed pseudogenes in the genome of Thoroughbred horses: Possibility of gene‐doping detection considering the presence of pseudogenes

et al. 2022

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Processed pseudogenes, also known as retrocopy genes, are copies of messenger RNAs that have been reverse transcribed into DNA and inserted into the genome. In this study, we identified 62 processed pseudogene candidates as intron‐less genes from whole‐genome sequencing (WGS) data of Thoroughbred horses using delly structural variation software. The 62 processed pseudogene candidates were confirmed by PCR amplification of intron‐less products. A total of 11 processed pseudogenes were confirmed in the genome of all 23 analysed horses, whereas three processed pseudogenes with structures of ATP11B, DPH3 and RPL17 were detected in only one of 115 horses by PCR amplification of intron‐less products. Currently, most of the gene doping tests proposed in human and horse sports are adapted PCR‐based methods using hydrolysis probes to detect exon/exon junctions in transgenes because the operation is simple and economical. However, when the pseudogene is present in the host genome, the PCR‐based methods may have a potential risk of detecting false positives. In this study, because processed pseudogenes that exist less frequently in the horse genome may affect PCR‐based transgene detection in gene‐doping tests, we propose and demonstrate that PCR amplification and sequencing using primers designed on transgene and promotors and/or polyadenylation signal for gene expression are useful for gene‐doping detection as an additional confirmatory test to prevent false positives.

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Section: Introductionmentioning

confidence: 99%

Identification of processed pseudogenes in the genome of Thoroughbred horses: Possibility of gene‐doping detection considering the presence of pseudogenes

et al. 2022

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“…As per the WADA guidelines for gene-doping tests, 23 PCR-based detection methods have several advantages, including a simple procedure, high sensitivity and high accuracy for specific targets. [6][7][8] However, when detecting low-copy numbers, it may be difficult to distinguish between positive and negative samples due to contamination, the presence of PCR inhibitors and substitution of bases at the primer and probe sites. 16,17,24 Digital PCR is more robust against PCR inhibitors and substitutions of template DNA than real-time PCR.…”

Section: Discussionmentioning

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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“…2019; Haughan et al . 2020). Recently, we developed a method for non‐targeted transgene detection, which detects intron‐less transgenes (cDNA form), using WGS data (Tozaki et al .…”

Section: Gene Targeted Intron Numbers Detected Introns 150 Bp Paired‐...mentioning

confidence: 99%

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

et al. 2021

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Summary Gene doping is prohibited in horseracing. In a previous study, we developed a method for non‐targeted transgene detection using DELLY, which is based on split‐read (SR) and paired‐end (PE) algorithms to detect structural variants, on WGS data. In this study, we validated the detection sensitivity of DELLY using artificially generated sequence data of 12 target genes. With DELLY, at least one intron was detected as a deletion in eight targeted genes using the 150 bp PE read WGS data, whereas all targeted genes were detected by DELLY using the 100 bp PE read data. The detection sensitivity was higher in 100 bp PE reads than in 150 bp PE reads, despite a lower total sequence coverage, probably because of mismatch tolerance between the mapped reads and reference genome. In addition, it was observed that the average intron size detected by SR alone was 293 bp and that that detected by both SR and PE was 8924 bp. Thus, we showed that transgenes with various intron–exon structures could be detected using DELLY, suggesting its application in gene‐doping control in horses.

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

Cited by 23 publications

References 33 publications

Identification of processed pseudogenes in the genome of Thoroughbred horses: Possibility of gene‐doping detection considering the presence of pseudogenes

Identification of processed pseudogenes in the genome of Thoroughbred horses: Possibility of gene‐doping detection considering the presence of pseudogenes

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

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

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