Ultra-sensitive and specific detection of porcine endogenous retrovirus (PERV) using a sequence-capture real-time PCR...

Use of porcine xenografts presents as a possible solution to the current shortage of human allografts limiting transplantation procedures. While no definitive observation of in vivo porcine endogenous retrovirus (PERV) transmission in humans has been reported, the in vitro ability of PERV to infect human cells and the observation of PERV transmission to immunodeficient mice suggest a need for ultra-sensitive techniques to monitor porcine xenograft recipients and contacts for possible PERV transmission. In an effort to enhance current PCR-based PERV detection, the feasibility of combining nucleic acid sequence-capture with use of a quantitative real-time 5' nuclease assay was examined. Sequence-capture by means of oligonucleotide hybridization to a conserved PERV gag sequence and attachment to magnetic beads was used to extract and concentrate PERV A, B and C DNA from sample material containing high levels of background human DNA. Sequence-capture oligonucleotide design incorporated selective substitution of dUTP for dTTP in order to facilitate eventual oligonucleotide destruction. In addition, sequence-capture oligonucleotides were located outside of the amplified region in order to minimize the effects of possible PCR carry-over. Quantitative PCR was then undertaken using a real-time 5' nuclease assay incorporating primers and probe also specific for a conserved PERV gag region. Sequence-capture real-time PCR assessment of PERV levels demonstrated a dynamic range of at least five orders of magnitude, a sensitivity between 0.005 and 0.028 PERV copies per microg background human DNA and a specificity between 98.2 and 100% (95% CI). In contrast, while real-time PERV PCR in the absence of a sequence-capture step demonstrated a similar specificity between 98.4 and 100% (95% CI), the sensitivity of this conventional approach was between 0.2 and 1.0 PERV copies per microg background human DNA. In conclusion, the increased sensitivity of PERV detection obtained by the combined use of PERV-specific sequence-capture and quantitative real-time PERV PCR suggest that this approach should enhance the effectiveness and reliability of monitoring procedures currently applied to porcine xenograft recipients and contacts.