QUANTITATIVE PCR FOR HUMAN HERPESVIRUS-6 AND HERPESVIRUS-7

A quantitative PCR assay for the detection of human herpesvirus 6 (HHV -6) (variants A and B) and HHV-7 DNAs in clinical samples was develope d. The assay uses a nonhomologous internal standard (IS) for each viru s that is coamplified with the wild-type target sequence in the same v ial and with the same pair of primers. This method allows for a correc tion of the variability of efficiency of the PCR technique. A standard curve is constructed for each experiment by coamplification of known quantities of the cloned HHV-6 or HHV-7 target templates with the resp ective IS. Absolute quantitation of the test samples is then achieved by determining the viral target/IS ratio of the hybridization signals of the amplification products and plotting this value against the stan dard curve. Using this assay, we quantitated the amount of HHV-6 or HH V-7 DNA in infected cell cultures and demonstrated an inhibitory effec t of phosphonoformic acid on the replication of HHV-6 and HHV-7 in vit ro. As the first clinical application of this procedure, we performed preliminary measurements of the loads of HHV-6 and HHV-7 in lymph node s from patients with Hodgkin's disease and AIDS. Application of this q uantitative PCR method should be helpful for elucidating the pathogeni c roles of HHV-6 and HHV-7.