Multiplex Detection of Transgenes Using Code Technology for Gene Doping Control

To ensure fair competition and sports integrity, genedoping isprohibited in horseracing and equine sports. One gene doping methodis by administering exogenous genes, called transgenes, to postnatalanimals. Although several transgene detection methods have been developedfor horses, many are unsuitable for multiplex detection. In this proof-of-conceptstudy, we developed a highly sensitive and multiplex transgene detectionmethod using multiple & pi;Code with identification patterns printedon the surface. The following steps were employed: (1) multiplex polymerasechain reaction amplification of 12 targeted transgenes in a singletube, (2) detection using a mixture of 12 probes labeled with different & pi;Codes, and (3) median fluorescence intensity measurement offluorescent & pi;Codes. Twelve transgenes cloned into plasmid vectorswere targeted, and 1500 copies of each plasmid were spiked into 1.5mL of horse plasma. Subsequently, a novel method using & pi;Codesucceeded in detecting all the transgenes using their DNA extracts.Additionally, we detected the erythropoietin (EPO) transgene in blood samples from a horse administered solely withthe EPO transgene using this method. Therefore, the & pi;Code detection method is considered suitable for multitargetgene detection in gene doping tests.