Design and storage stability of reference materials for microfluidic quantitative PCR-based equine gene doping tests.
Abstract: One method of gene doping in horseracing is administering of exogenous genetic materials, known as transgenes. Several polymerase chain reaction (PCR)-based methods have been developed for detecting transgenes with high sensitivity and specificity. However, novel designs for reference materials (RMs) and/or positive template controls (PTCs) are necessary for simultaneous analysis of multiple transgene targets. In this study, we designed and developed a novel RM for simultaneously detecting multiple targets via microfluidic quantitative PCR (MFQPCR). Twelve equine genes were selected as targets in this study. A sequence region including primers and probes for quantitative PCR was designed, and a 10 bp sequence was inserted to allow the RM to be distinguished from the original transgene sequences. The sequences of individual detection sites were then connected for 12 genes and cloned into a single plasmid vector. We performed fragment size analysis to distinguish between the PCR products of the original transgene sequence and those of the RM, enabling identification of RM contamination. PTCs diluted to 10,000, 1,000, 100, and 10 copies/µl with horse genomic DNA from RM were stably stored at 4°C for 1 year. As digital PCR enabled absolute quantification, the designed substances can serve as an RM. These findings indicate that the RM design and storage conditions were suitable for gene doping tests using MFQPCR.
©2021 The Japanese Society of Equine Science.
Publication Date: 2021-12-28 PubMed ID: 35023990PubMed Central: PMC8731687DOI: 10.1294/jes.32.125Google Scholar: Lookup
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Summary
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This study focused on improving methods to detect the use of genetically engineered materials, often used as performance enhancers, in horse racing by developing a reference material capable of identifying multiple transgenes simultaneously through microfluidic quantitative polymerase chain reaction (PCR).
Research Overview
- The main aim of this research was to create a reference material (RM) that can simultaneously detect multiple transgenes in horses. Gene doping in horseracing is often done through administering foreign, genetically-engineered materials known as transgenes.
- Polymerase chain reaction or PCR-based methods have previously been used to detect these transgenes due to their high sensitivity and specificity, but there are gaps in their ability to analyze multiple transgene targets at once.
- This led to the creation of a novel RM for microfluidic quantitative PCR (MFQPCR) which was designed to handle detection of different multiple targets concurrently.
Choosing Gene Targets
- Twelve equine genes were selected as targets for the study and a sequence region that includes primers and probes for quantitative PCR was designed for each gene.
- A 10 base pair sequence was inserted to allow the RM to be distinguishable from the original transgene sequences, essentially creating a signature unique to the RM.
Development of the Reference Material
- The sequences of individual detection sites of the 12 genes were then connected and cloned into a single plasmid vector, creating a multi-purpose testing material.
- To avoid potential contamination of the test samples with the reference material, fragment size analysis was performed. This made it possible to identify RM contamination as PCR products of the RM and the original transgene sequence would be different in size.
Storing and Quantifying the Reference Material
- The researchers also explored storage solutions for the RM, with positive template controls (PTCs) diluted with horse genomic DNA from RM stored at 4°C for 1 year proving to be stable.
- Digital PCR was used to perform absolute quantification, confirming that the created substances could serve as an RM.
In conclusion, the study resulted in the creation of a usable RM that can simultaneously detect and analyze multiple transgenes in horse samples for detecting dopant use in horse racing. The RM developed, along with the storage solution, will aid in maintaining integrity and fairness in the sport.
Cite This Article
APA
Tozaki T, Ohnuma A, Kikuchi M, Ishige T, Kakoi H, Hirota KI, Kusano K, Nagata SI.
(2021).
Design and storage stability of reference materials for microfluidic quantitative PCR-based equine gene doping tests.
J Equine Sci, 32(4), 125-134.
https://doi.org/10.1294/jes.32.125 Publication
Researcher Affiliations
- Genetic Analysis Department, Laboratory of Racing Chemistry, Tochigi 320-0851, Japan.
- Genetic Analysis Department, Laboratory of Racing Chemistry, Tochigi 320-0851, Japan.
- Genetic Analysis Department, Laboratory of Racing Chemistry, Tochigi 320-0851, Japan.
- Genetic Analysis Department, Laboratory of Racing Chemistry, Tochigi 320-0851, Japan.
- Genetic Analysis Department, Laboratory of Racing Chemistry, Tochigi 320-0851, Japan.
- Genetic Analysis Department, Laboratory of Racing Chemistry, Tochigi 320-0851, Japan.
- Equine Department, Japan Racing Association, Tokyo 106-8401, Japan.
- Genetic Analysis Department, Laboratory of Racing Chemistry, Tochigi 320-0851, Japan.
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Citations
This article has been cited 1 times.- Maniego J, Harding C, Habershon-Butcher J, Hincks P, Ryder E. Administration and detection of a multi-target rAAV gene doping vector in horses using multiple matrices and molecular techniques. Gene Ther 2024 Sep;31(9-10):477-488.
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