The incidence of porcine endogenous retrovirus (PERV) infecting human cells in vitro has heightened safety concerns over the transmission of PERV to xenograft recipients, making the establishment of diagnostic tests to closely monitor PERV of priority concern. This study detects PERV pol and gag sequences by designing highly specific polymerase chain reaction (PCR) assays with conserved primers. Three sets of primers for the env region are designed to identify A, B, and C subtypes of PERV, and a PCR assay is developed to detect a specific mitochondria sequence as a pig cell marker. These tests allow clinicians to effectively monitor PERV transmission in the serum, cell, or excretion fluid associated with the use of porcine source materials.