Quantitative, Fluorogenic Probe PCR Assay for Detection of Human Herpesvirus 8 DNA in Clinical Specimens

Abstract

A quantitative, fluorescence-based PCR assay (TaqMan-based system) was developed for detection of human herpesvirus 8 (HHV-8) DNA in clinical specimens. Primers and probes chosen from each of five 10-kb segments from the unique 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. Although several of the primer-probe sets performed similarly with BCBL-1 DNA that had been diluted in water, their performance differed when target DNA was diluted in a constant background of uninfected cell DNA, an environment more relevant to their intended use. The two best primer-probe combinations were specific for HHV-8 relative to the other known human herpesviruses and herpesvirus saimiri, a closely related gammaherpesvirus of nonhuman primates. PCRs included an enzymatic digestion step to eliminate PCR carryover and an exogenous internal positive control that enabled discrimination of false-negative from true-negative reactions. The new assays were compared to conventional PCR assays for clinical specimens (saliva, rectal brushings, rectal swab specimens, peripheral blood lymphocytes, semen, and urine) from 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.