Polymerase chain reaction assays for the diagnosis of infection with the porcine endogenous retrovirus and the detection of pig cells in human and nonhuman recipients of pig xenografts
Wm. Switzer et al., Polymerase chain reaction assays for the diagnosis of infection with the porcine endogenous retrovirus and the detection of pig cells in human and nonhuman recipients of pig xenografts, TRANSPLANT, 68(2), 1999, pp. 183-188
Background. Pigs offer an unlimited source of xenografts for humans. Howeve
r, recipients of pig xenografts are inevitably exposed to the porcine endog
enous retrovirus (PERV), which is carried in the pig germline, The ability
of PERV to infect human cells in vitro has heightened safety concerns regar
ding the transmission of PERV to pig xenograft recipients.
Methods. In response to the need to establish laboratory tests for the surv
eillance of PERV infection, we have developed polymerase chain reaction (PC
R) assays to detect PERV pol and gag sequences by using conserved primers a
nd probes. In addition, we have developed a PCR assay to detect pig-specifi
c mitochondrial DNA (mtDNA) sequences as a marker of pig cells.
Results. Analysis of assay sensitivities using cloned target copies in a ba
ckground of human DNA demonstrated a detection threshold of 1, 5, and 1 cop
y for the PERV gag, pol, and pig mtDNA PCR assays, respectively, All three
PCR assays gave negative results on peripheral blood lymphocyte samples fro
m 69 humans, as well as 6 baboons and 6 macaques, demonstrating 100% specif
icity. The PERV and pig mtDNA assays were integrated into a simple testing
algorithm that allows the differentiation between pig cell microchimerism a
nd true xenogeneic infection, To allow for monitoring of PERV expression, a
reverse transcriptase-PCR assay was also developed to detect cell-free PER
V RNA.
Conclusion. The use of the diagnostic tests described here will help define
the risks of PERV transmission associated with the use of pig xenografts i
n humans and nonhuman primates.