Emerging wound-healing injectable polydeoxyribonucleotide: potential as a prohibited doping method and its simple detection via CRISPR/ Cas12a system

Abstract

Polydeoxyribonucleotide (PDRN), derived from chum salmon (Oncorhynchus keta), is a mixture of hydrolyzed DNA fragments used in various clinical applications. Its therapeutic value stems from its ability to promote wound healing by upregulating growth factors like VEGF, FGF, and HIF-1. However, PDRN's regenerative properties raise concerns about its potential misuse in sports. Studies suggest it may enhance athletic performance by stimulating muscle growth, recovery, and endurance through mechanisms such as satellite cell activation, angiogenesis, and anti-inflammatory effects. These potential performance-enhancing effects could be considered gene or cell doping, prohibited by the World Anti-Doping Agency (WADA). To address this concern, we developed a sensitive and specific detection method for PDRN misuse based on the CRISPR-Cas12a system. This method targets conserved 12S and 16S rDNA sequences unique to salmonids. A direct PCR method was optimized to amplify these target sequences from human plasma and urine without prior DNA extraction. The amplified DNA was then subjected to Cas12a-mediated detection, resulting in a fluorescent signal upon successful target recognition. This method demonstrated high sensitivity, detecting as little as 0.8 pg (0.3 genome copies) of O. keta DNA in 10 mu L of biological samples within 90 min, surpassing the detection limits of many current doping agents.