Optimization and implementation of four duplex quantitative polymerase chain reaction assays for gene doping control in horseracing

Abstract

The concern about gene doping has remained high in horseracing and other equestrian competitions. Our laboratory has previously developed a duplex quantitative polymerase chain reaction (qPCR) assay capable of detecting in equine blood the human erythropoietin (hEPO) transgene and equine tubulin α 4a (TUBA4A) gene as an internal control the latter providing quality control over DNA extraction and qPCR. This study aimed to optimize the method for routine testing of regulatory samples. The use of an automated DNA extraction system has increased the sample throughput, consistency of DNA extraction, and recovery of reference materials. The use of reduced concentration of primers and hydrolysis probe for internal control minimized their competition with transgene amplification and improved the assay sensitivity. Spike‐in of an exogenous internal control at low concentration for plasma analysis has also been validated. Using the new workflow, four duplex qPCR assays have been developed for the detection of transgenes, namely, hEPO, human growth hormone (hGH), insulin‐like growth factor 1 (hIGF‐1), and equine EPO (eEPO). The estimated limits of detection (LODs) of each transgene were 2000 copies/mL of blood and 200 copies/mL of plasma. This method could detect the presence of transgene in blood and plasma collected from a horse administered intramuscularly (IM) with recombinant adeno‐associated virus (rAAV) carrying the hEPO transgene. A longer detection time was observed in blood than in plasma. The methods have been applied to the screening of over a thousand official racehorse samples since June 2020 for the presence of these transgenes.

A multiplex qPCR assay for transgenes detection: A novel approach for gene doping control in horseracing using conventional laboratory setup

“…with satisfactory recovery ($91.7%). 13 The extracted DNA was then subjected to downstream multiplex and duplex qPCR assays.…”

“…As a result, blood represents the sample of choice for transgene(s) detection. 13,14 Doping control laboratories worldwide have been actively developing methods for transgene(s) detection. The exon-exon junction(s) of a transgene represents the signature(s) of its exogenous origin and has been the 'target(s)' for transgene detection.…”

“…Approaches based on either polymerase chain reaction (PCR) or sequencing have been reported for detecting transgenes in equine biological specimens. [13][14][15][16][17][18][19][20] Among them, PCR-based methods (e.g., qPCR and dPCR) are considered a mature technology in the context of doping control and have resulted in the publication of 'Laboratory Guidelines -Gene Doping Detection based on Polymerase Chain Reaction (PCR)' by the World Anti-Doping Agency 21 and 'Guidelines for the Minimum Criteria for Identification of Transgenes or Vectors by Polymerase Chain Reaction (PCR) Analysis' by the Association of Official Racing Chemists. 22 Hundreds of potential performance-enhancing genes have already been proposed.…”

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

“…Many methods to detect inserted transgenes using quantitative PCR have been developed for gene-doping control [ 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 ]. By designing hydrolysis probes targeting the exon/exon junction, transgenes can be detected in a target-specific manner.…”

Multiplex Detection of Transgenes Using πCode Technology for Gene Doping Control

“…One gene doping method is to administer exogenous genes, called transgenes, to postnatal animals as an abuse and/or misuse of gene therapy agents . Various detection methods for transgenes have been developed for horses, such as real-time polymerase chain reaction (PCR), − digital PCR, , and next-generation sequencing (NGS). − For the specific detection of targeted transgenes, hydrolysis probes designed at the exon/exon junction were used for quantitative PCR detection. The WADA describes laboratory guidelines for PCR-based transgene detection…”