Quantitative analysis of Epstein - Barr virus DNA load in bone marrow transplant recipients by using real-time PCR

ABSTRACT

Quantification of Epstein - Barr virus [EBV] in peripheral blood mononuclear cells [PBMNC] of allogenic bone marrow transplant [BMT] recipients is important because EBV-associated posttransplant Lymphoproliferative disease [PTLD] can occur after transplantation due to immunosup-pression therapy. To this end we chose Real-Time PCR using TaqMan probe. For the standard curve, we cloned BALF5 gene of EBV into a plasmid vector. After purification of the EBV-clone and calculation of plasmid copy number, the standard curve was constructed by using serial dilution of the plasmid clone. We were able to detect from 2 to 107 copies per 2x105 PBMNC with wide linear range. The mean EBV DNA copy number was 103.7 copies per 2x105 PBMNC. In this study, No patient of 35 BMT recipients [275 PBMNC samples] developed PTLD during five months follow up post transplant. EBV copy numbers in 22 samples [3 patients] out of 35 BMT recipients were higher than cut off value with symptoms like fever and pulomonary noddes [9%]. The virus load in one patient in the last sample obtained was 72400 copies. We detected low levels of EBV DNA in 20 BMT patients [57/1%]. Real-Time PCR is useful to measure virus load in PBMNC. Detection of EBV in PBMNC samples may be valuable predictive marker to prognosis PTLD. Further studies need to determine ac-curate viral cut off value for treatment patients at risk for PTLD