To ensure fair competition and sports integrity, gene
doping is
prohibited in horseracing and equine sports. One gene doping method
is by administering exogenous genes, called transgenes, to postnatal
animals. Although several transgene detection methods have been developed
for horses, many are unsuitable for multiplex detection. In this proof-of-concept
study, we developed a highly sensitive and multiplex transgene detection
method using multiple πCode with identification patterns printed
on the surface. The following steps were employed: (1) multiplex polymerase
chain reaction amplification of 12 targeted transgenes in a single
tube, (2) detection using a mixture of 12 probes labeled with different
πCodes, and (3) median fluorescence intensity measurement of
fluorescent πCodes. Twelve transgenes cloned into plasmid vectors
were targeted, and 1500 copies of each plasmid were spiked into 1.5
mL of horse plasma. Subsequently, a novel method using πCode
succeeded in detecting all the transgenes using their DNA extracts.
Additionally, we detected the erythropoietin (EPO) transgene in blood samples from a horse administered solely with
the EPO transgene using this method. Therefore, the
πCode detection method is considered suitable for multitarget
gene detection in gene doping tests.