Performance characteristics of two real-time PCR assays for the quantification of Epstein-Barr virus DNA

We compared the performance of a laboratory-developed 5'-nuclease real-time polymerase chain reaction assay and a commercial assay (LightCycler, Roche Diagnostics, Indianapolis, IN) for quantification of Epstein-Barr virus (EBV) DNA. Using standards provided by the manufacturer, the LightCycler assay was linear from 100 to 1 million copies per reaction. Based on dilution of a plasmid containing the amplicon, the laboratory-developed assay was linear-from 22 to 45 million copies per reaction. Both assays detected 0.5 copies of genomic EBV DNA per reaction; both, showed good reproducibility with coefficients of variation front 0.3% to 2.4% for the LightCycler and 1.8% to 5.1% for the laboratory-developed assay. For 31 whole blood specimens with measurable values in both assays, the viral load values obtained with the LightCycler averaged 2.3 fold higher than those obtained with the laboratory-developed assay. Testing 30 matched whole blood and plasma samples in the laboratory-developed assay showed whole blood viral load values averaged 10 fold higher than those for plasma. The LightCycler and laboratory-developed assays are sensitive and reproducible with broad linear ranges. Further clinical evaluation is needed to determine the viral load cutoff that is predictive of posttransplantation lymphoproliferative disorders.