Quantitative, fluorogenic probe PCR assay for detection of human herpesvirus 8 DNA in clinical specimens

A quantitative, fluorescence-based PCR assay (TaqMan-based system) was deve loped for detection of human herpesvirus 8 (HHV-8) DNA in clinical specimen s. Primers and probes chosen from each of five 10-kb segments from the uniq ue region of the HHV-8 genome were evaluated for sensitivity with dilution series of DNA extracted from a cell line (BCBL-1) that harbors HHV-8 DNA. A lthough several of the primer-probe sets performed similarly,,vith BCBL-1 D NA that had been diluted in water, their performance differed when target D NA was diluted in a constant background of uninfected cell DNA, an environm ent more relevant to their intended use. The two best primer-probe combinat ions were specific for HHV-8 relative to the other known human herpesviruse s and herpesvirus saimiri, a closely related gammaherpesvirus of nonhuman p rimates. PCRs included an enzymatic digestion step to eliminate PCR carryov er and an exogenous internal positive control that enabled discrimination o f false-negative from true-negative reactions. The new assays were compared to conventional PCR assays for clinical specimens (saliva, rectal brushing s, rectal swab specimens, peripheral blood lymphocytes, semen, and urine) f rom human immunodeficiency virus-positive patients with or without Kaposi's sarcoma. In all instances, the new assays agreed with each other and with the conventional PCR system. In addition, the quantitative results obtained with the new assays were in good agreement both for duplicate reactions in the same assay and between assays.