Normalized quantification by real-time PCR of Epstein-Barr virus load in patients at risk for posttransplant lymphoproliferative disorders

The load of Epstein-Barr virus (EBV) in peripheral blood mononuclear cells of transplant recipients represents a predictive parameter for posttranspla nt lymphoproliferative disorders (PTLD). The aim of our work was to develop a rapid and reliable PCR protocol for the quantification of cell-associate d EBV DNA in transplant recipients. In contrast to previous studies, a prot ocol that facilitated quantification independent of photometric nucleic aci d analysis was established. We took advantage of the real-time PCR technolo gy which allows for single-tube coamplification of EBV and genomic C-reacti ve protein (CRP) DNA. EBV copy numbers were normalized by division by the a mount of CRP DNA, with the quotient representing the actual amount of ampli fiable genomic DNA per reaction. Coamplification of CRP DNA did not result in a diminished detection limit for EBV, By using the protocol without norm alization, EBV copy numbers in 4 out of 10 PTLD patients were within the no rmal range determined with data for 114 transplant recipients that served a s controls. After normalization, however, all of the PTLD patients had a hi gher viral load than the control population, indicating an increased sensit ivity of the assay. Moreover, EBV copy numbers obtained for one patient by conventional quantification and suggestive of relapsing PTLD were within no rmal range after normalization. We conclude that normalization of PCR signa ls to coamplified genomic DNA allows a more accurate quantification of cell -bound EBV.