Monitoring of Epstein-Barr virus DNA load in peripheral blood by quantitative competitive PCR

A competitive quantitative PCR (Q-PCR) assay combined with simple silica-ba sed DNA extraction was developed for monitoring of Epstein-Barr virus (EBV) DNA load in unfractionated peripheral blood. The Q-PCR is based on competi tive coamplification of a highly conserved 213-bp region of the EBNA-1 open reading frame with an internal standard (IS), added in a known concentrati on. The IS has the same amplicon length and base composition as the wild-ty pe (WT) EBNA-1 amplicon but differs in 23 internally randomized bases. Comp etitive coamplification yields two PCR products that are quantified by enzy me immunoassay or by electrochemiluminescence detection, with probes specif ic for the 23 differing internal nucleotides, The Q-PCR has a sensitivity o f 10 copies of either WT or IS plasmid DNA. The Q-PCR was validated by quan tification of known amounts of plasmid containing the WT EBNA-1 target. Fur thermore, we determined EBV genome copy numbers in different cell lines. Fo r EBV quantification in clinical samples, DNA was isolated hom lysed whole blood by silica-affinity purification. Forty-six percent of healthy donor p eripheral blood samples were positive by Q-PCR. In most of these samples, v iral load was less than 2,000 EBV copies/ml of blood. In peripheral blood s amples from two AIDS-related non-Hodgkin's lymphoma patients, elevated EBV loads (up to 120,000 copies/ml) were observed, which decreased upon therapy . In Burkitt's lymphoma patients, up to 4,592,000 EBV genome copies/ml of b lood were detected. In conclusion, the EBNA-1-based Q-PCR assay provides a reproducible, accurate, and easy method for studying the relationship betwe en EBV load and clinical parameters.