Multiplex detection of seven transgenes for human gene doping analysis

Abstract

Abstract Gene doping is known as the manipulation of congenital traits by gene therapeutic approaches with the intent of illicit athletic performance enhancement. A panel prototype suitable for multiplex gene doping detection by combining multiplex Polymerase Chain Reaction (PCR)-amplification with Matrix-Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) analysis was developed and examined for its specificity and sensitivity, and its applicability in human sports drug testing programs was assessed. The panel comprises 20 assays for exon-exon-junction detection of seven human transgenes (EPO, FST, GH1, IGF1, MSTN (propeptide), VEGFA, VEGFD), which have been considered as material to routine doping controls, in one reaction. Alongside, a suitable reference material (RM) was designed and tested for its utility. An estimated LOD95 of 1,500 cp / mL or 30 copies (cp) per reaction of the panel and 500 cp / mL or 10 cp per reaction of the RM was determined in plasmid-spiked human whole blood samples. The specificity and applicability of the panel and the RM was further determined by testing equine plasma samples obtained from an animal that received rAAV-delivered human transgenic EPO as well as 111 native human doping control samples.