Validation Study of the High-throughput Gene Doping Assay (HiGDA) Platform for exogenous EPO Gene Doping in Human Blood

Abstract

Nowadays, advancements in gene therapy have propelled the emergence of a novel doping method known as gene doping. To curb these potentially harmful and unpredictable practices, the development of an efficient, sensitive testing method to detect doping genes is critical. We present a novel high-throughput gene doping assay (HiGDA) that utilizes Cas12a coupled with blood direct PCR. This innovative approach enables direct amplification of exogenous EPO gene from whole blood samples of athletes. Furthermore, Cas12a, a protein capable of sequence-specific DNA recognition and providing fluorescent response, is incorporated to screen for the presence of doping genes within an assay. We validated HiGDA using 20 whole blood samples spiked with exogenous EPO gene, including samples containing 10 copies of EPO gene and appropriate control samples, according to the World Anti-Doping Agency's (WADA) guidelines for gene doping detection. The direct amplification of the exogenous EPO gene in whole blood samples was performed followed by an initial testing procedure (ITP) using gel electrophoresis within 20 minutes. The ITP successfully detected all samples spiked with 10 copies of the target EPO genes and even 19 out of 20 samples containing 5 copies. Subsequently, a confirmation procedure (CP) employing Cas12a fluorescence response and targeting a different exon-exon junction from the ITP was conducted. This CP, taking 30 minutes, achieved a 100% detection rate in samples spiked with 10 copies and 95% for 5 copies. Notably, the HiGDA demonstrated no false-positive or false-negative results, verifying its high accuracy, sensitivity, specificity, and simplicity.