Quantitative real time PCR assay for detecting EBV virus in multiple sample types.

We are developing a real time quantitative PCR assay to detect EBV in serum, plasma, whole blood, tissue and spinal fluid. Real time PCR, with its intrinsic quantitative capacity, is an excellent method for measuring EBV viral load. Epstein Barr virus is a member of the Herpesvirus family, with a tropism for B lymphocytes, where it establishes latency. In transplant settings, it causes post transplantation lymphoproliferative isorder (PTLD). High doses of immunosuppressive drugs allow the virus to escape the immune system, which normally keeps the latent virus in check. Symptoms of PTLD can mimic those of organ rejection, leading to increased immunosuppression, when a decrease in dosage is actually necessary. The primers and probes for this assay are supplied by Epoch/Nanogen and target a region of the BNFR1 gene. This EBV assay design has the probe-binding site overlapping one of the primer binding sites by five nucleotides. Dilutions of a plasmid containing the cloned amplicon are used as standards. A new standard curve is generated and stored with each new lot of EBV reagents or Taq polymerase. Sequestration of reagent lots and the lyophilization of control material have been shown to help maintain stability of assay performance over the period that a stored standard curve is being used. A plasmid internal control is included in the sample extraction to detect PCR inhibition and extraction failures.