Current gene-doping detection strategies primarily focus on identifying transgenes introduced via viral or plasmid vectors, targeting exon–exon junctions using hydrolysis probe-based quantitative PCR. These DNA-centered methods exhibit high specificity and sensitivity. However, recent advances in biopharmaceuticals have accelerated the development of RNA-based therapeutics, which bypass transcription and initiate protein translation directly. This leads to enhanced expression efficiency and reduced risk of genomic integration, making mRNA drugs an attractive modality — including potential misuse in gene doping. In this study, we address the challenge of detecting RNA-based gene doping agents, which traditionally require labor-intensive RNA extraction and reverse transcription steps. We report the development of direct-SATORI, a novel amplification-free detection platform employing CRISPR-Cas13a in conjunction with a femtoliter-scale microchamber array. This system enables direct, single-molecule-level detection of target RNAs from biological matrices without prior nucleic acid extraction or cDNA synthesis. As a proof of concept, a synthetic equine erythropoietin mRNA construct was used to simulate RNA-based doping. Method validation was performed using plasma samples spiked with the equine erythropoietin N1-methylpseudouridine modified mRNA encapsulated within lipid nanoparticles. Furthermore, we successfully detected the administered modified mRNA/LNP in post-injection plasma samples from a treated horse, demonstrating the feasibility of gene-doping detection. This platform, direct-SATORI, offers a promising tool for future anti-doping efforts, particularly as RNA-based modalities become increasingly prevalent in both therapeutic and illicit contexts.
In addition, we estimated the spontaneous mutation rate in Thoroughbred horses. Whole-genome sequencing of a Thoroughbred parent-offspring trio revealed 46 single nucleotide variants and 2 deletions violating Mendelian inheritance. This corresponds to a de novo mutation rate of 1.92 × 10⁻⁸ per generation. This estimate may provide a critical reference for distinguishing gene edits from spontaneous mutations in equine gene-editing test.
Together, these findings contribute to the establishment of robust frameworks for gene-doping control in horse racing.