Low-copy transgene detection using nested digital PCR for gene doping control.

Gene doping is prohibited for fair competition in human and horse sports. One style of gene doping is the administration of an exogeneous gene, called a transgene, to postnatal humans and horses. Although many transgene detection methods based on quantitative PCR, including real-time PCR and digital PCR, have been recently developed, it remains difficult to reliably detect low-copy transgenes. In this study, we developed and validated a nested digital PCR method to specifically detect low-copy transgenes. The nested digital PCR consists of 1) pre-amplification using conventional PCR and 2) droplet digital PCR detection using a hydrolysis probe. Using 5, 10, 20, 60, and 120 transgene copies as template, 496.0, 1089.7, 1820.7, 4313.3, and 7840.0 copies/μL respectively were detected using our nested digital PCR. Although high concentrations of phenol, proteinase K, ethanol, EDTA, heparin, and genomic DNA all inhibited pre-amplification, their effects on the digital PCR detection were limited. Once pre-amplification was successful, even substitution of bases within the primers and probes had minimal effects on transgene detection. The nested digital PCR developed in this study successfully detected low-copy transgenes and can be used to perform a qualitative test, indicating its usefulness in the prevention of false positives and false negatives in gene doping detection.