Codon changes challenge PCR-based gene doping detection.

Genetic/genomic manipulation techniques (gene transfer/delivery, gene editing, etc.) have become more and more mature, and the illegal use as gene doping in sports has drawn attentions. World Anti-Doping Agency (WADA) strictly prohibits gene doping, and has issued guideline on quantitative real-time PCR (qPCR) detections. However, the technical feature of qPCR makes it difficult to detect new doping targets, and codon changes on targets may also affect detection efficiency. Here, we prepare standard materials for genomic and transgenic versions of human EPO (hEPO) gene, and design qPCR primers to check the consequences of codon changes on gene doping detection. We confirm that carefully designed qPCR assays could indeed capture transgene signal, but codon changes on the transgene could severely undermine detection efficiency. We have also mimicked real world gene doping scenario by mixing genomic and transgenic versions of hEPO, and qPCR could detect wild-type but not codon-changed transgenes. As a method validation for such a challenge, we also use Sanger sequencing to confirm that sequencing could easily capture gene doping even for codon-changed transgenes. Our study confirms that codon changes will challenge qPCR-based gene doping detection, and calls for un-biased detection tools based on high-throughput sequencing in the future.