Development of a real-time quantitative assay for detection of Epstein-Barr virus.

With the use of real-time PCR, we developed and evaluated a rapid, sensitive, specific, and reproducible method for the detection of Epstein-Barr virus (EBV) DNA in plasma samples. This method allowed us to screen plasma and serum samples over a range between 100 and 10(7) copies of DNA per ml using two sample preparation methods based on absorption. A precision study yielded an average coefficient of variation for both methods of less than 12%, with a coefficient of regression for the standard curve of a minimum of 0. 98. We detected EBV DNA in 19.2% of plasma samples from immunosuppressed solid-organ transplant patients without symptoms of EBV infections with a mean load of 440 copies per ml. EBV DNA could be detected in all transplant patients diagnosed with posttransplant lymphoproliferative disorder, with a mean load of 544,570 copies per ml. No EBV DNA could be detected in healthy individuals in nonimmunosuppressed control groups and a mean of 6,400 copies per ml could be detected in patients with infectious mononucleosis. Further studies revealed that the inhibitory effect of heparinized plasma could be efficiently removed by use of an extraction method with Celite as the absorbent.