A calibrated quantitative real-time polymerase chain reaction (PCR) assay for the measurement of EBV DNA of both EBV-1 and 2 subtypes has been developed, combining the detection of the EBV DNA and a synthetic DNA calibrator in a multiplex PCR format.

Scientists at the San Raffaele Scientific Institute (Milan, Italy) developed a real time quantitative PCR assay and clinically validated the method. Several cohorts of individuals including patients with EBV-associated diseases obtained from different geographic areas were recruited and tested. The clinical sensitivity and specificity of the system were evaluated by testing 181 peripheral blood mononuclear cell (PBMCs) and plasma specimens obtained from 21 patients subjected to bone marrow transplantation, 70 HIV-seropositive subjects, and 23 healthy controls.

Optimization of the reactions was achieved by determining the concentrations of primers and probes, as well as the annealing temperature yielding the highest intensity of reporter fluorescent signal without a reduction in specificity or sensitivity. This was achieved using the Absolute qPCR ROX master mix (ABgene; Epsom, UK). The assay displays a wide dynamic range and a high degree of accuracy even in the presence of 1 μg of human genomic DNA. This assay measures with the same efficiency EBV DNA from strains prevalent in different geographic areas. Patients affected by EBV-associated posttransplant lymphoproliferative disorders had the highest frequency of EBV detection and the highest viral load. Persons infected with HIV had higher levels of EBV DNA load in PBMCs and a higher frequency of EBV plasma viremia compared to healthy controls.

The authors concluded that this new assay provides a reliable high-throughput method for the quantitation of EBV DNA in clinical samples. The calibrated real-time PCR technique will offer clinicians a fast and reliable method for establishing an etiologic link between EBV and human diseases, as well as to monitor the risk of developing EBV-associated malignancies and the efficacy of therapeutic approaches against EBV infection. The study was published in the December 2011 issue of the Journal of Virological Methods.

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