Screening for gene doping transgenes in horses via the use of massively parallel sequencing.
- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
The study explores a method of identifying gene doping in horses, using modern sequencing technology. It uses a two-step PCR method in parallel sequencing to detect multiple doping targets simultaneously, and tests its sensitivity through real-world applications.
Methodology
The researchers developed a screening method using a two-step Polymerase Chain Reaction (PCR) procedure. This is a DNA amplification technique that reproduces specific DNA sequences from the sample. Their method involves massively parallel sequencing, which allows them to screen for multiple doping targets at once. They pooled primer sets, sequences that start DNA replication, to provide scalability, using combinational indexing.
Quality Control and Data Processing
Reference standards with modified sequence were used as controls to ensure the accuracy and reliability of the experiment. They also employed custom software to generate transgene-specific amplicons, fragments of DNA or RNA that are produced by amplification, from any genome annotated in Ensembl, a database that provides DNA sequences and related information. This custom software allows for quick assay design. Additional scripts were used to process the data obtained from the experiments in batches. These scripts automatically filter sequences based on their quality and assign hits based on recognisable patterns.
Testing and Evaluation
To test this approach, they established a workflow with an initial set of 31 transgene and vector feature targets. A real-world setting was then simulated to evaluate the sensitivity of the parallel sequencing method. They administered an rAAV vector, a common gene therapy vector, intramuscularly into two horses and measured the detection sensitivity between this new method and real-time quantitative PCR (qPCR), a technique used to monitor the progress of the PCR reaction in real time. The vector was detected using both methods up to 79 hours post-administration, proving the potential of their new method for gene-doping detection.
Results
The results showed that the new method was effective in detecting the administered vector. However, detection became less frequent after 96 hours. This indicates that while the massively parallel sequencing PCR method can detect doping, it may be less effective for longer periods after administration. This should be considered when using this method for detection. However, it is a step forward in identifying gene doping in horses, providing a scalable and efficient means of testing.
Cite This Article
Publication
Researcher Affiliations
- Sport and Specialised Analytical Services, LGC, Fordham, Cambridgeshire, UK.
- Sport and Specialised Analytical Services, LGC, Fordham, Cambridgeshire, UK.
- British Horseracing Authority, London, UK.
- National Measurement Laboratory, LGC, Teddington, Middlesex, UK.
- School of Biosciences & Medicine, Faculty of Health & Medical Sciences, University of Surrey, Guildford, Surrey, UK.
- Sport and Specialised Analytical Services, LGC, Fordham, Cambridgeshire, UK.
- Sport and Specialised Analytical Services, LGC, Fordham, Cambridgeshire, UK.
- Sport and Specialised Analytical Services, LGC, Fordham, Cambridgeshire, UK. edward.ryder@lgcgroup.com.
MeSH Terms
- Animals
- Doping in Sports / methods
- Genetic Therapy
- High-Throughput Nucleotide Sequencing
- Horses
- Real-Time Polymerase Chain Reaction / methods
- Transgenes
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Citations
This article has been cited 2 times.- Lu Y, Yan J, Ou G, Fu L. A Review of Recent Progress in Drug Doping and Gene Doping Control Analysis.. Molecules 2023 Jul 18;28(14).
- Tozaki T, Ohnuma A, Nakamura K, Hano K, Takasu M, Takahashi Y, Tamura N, Sato F, Shimizu K, Kikuchi M, Ishige T, Kakoi H, Hirota KI, Hamilton NA, Nagata SI. Detection of Indiscriminate Genetic Manipulation in Thoroughbred Racehorses by Targeted Resequencing for Gene-Doping Control.. Genes (Basel) 2022 Sep 4;13(9).