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.Human herpesvirus 8 (HHV-8), also known as Kaposi's sarcoma (KS)-associated herpesvirus, is the etiologic agent of KS; it has been associated with a subset of body cavity lymphomas known as primary effusion lymphoma and multicentric Castleman's disease (reviewed in references 8, 13, and 15). Many aspects of HHV-8 natural history are unknown, especially with regard to viral load in various bodily fluids in relation to time after infection, immune function, and therapy directed either at the virus, the end-organ disease, or the immune system. Study of such relationships requires reproducible, accurate, and precise methods for determining DNA concentrations. Semiquantitative and robust quantitative systems have been described, but in general they are not easily adapted for high throughput and are susceptible to PCR contamination due to both the number of manipulations required and the need to manipulate amplified products to enable detection (2, 3, 6, 7).Methods have been developed for the detection of PCR products that enable continuous monitoring of product accumulation during PCR, e.g., TaqMan, LightCycler, fluorescence resonance energy transfer probes (1, 5), and molecular beacons (17); these methods are collectively referred to as realtime PCR. These methods do not require specimen manipulation after reaction assembly, thereby substantially reducing the possibility of PCR carryover contamination and reducing net labor. In addition, these systems can be adapted to yield accurate and precise quantitative inf...

Molecular Genetics of Kaposi's Sarcoma-Associated Herpesvirus (Human Herpesvirus 8) Epidemiology and Pathogenesis

“…Studies of three different populations with high STD and AIDS prevalences in San Francisco agree with this behavioral risk factor, suggesting that insertive oral sex may be the highest risk behavior, as inferred from the easy detection of KSHV in saliva but a low viral load in semen (386). In agreement, many studies have found KSHV in the saliva of seropositive patients (45,266,392,481,513), and KSHV has been detected in prostate tissue and the male urogenital tract (132,348,481,483); detection of KSHV in the ejaculate has been reported but remains controversial (255,300,348,396,481; P. Gupta, M. K. Singh, C. Rinaldo, M. Ding, H. Farzadegan, A. Saah, D. Hoover, P. Moore, and L. Kingsley, Letter, AIDS 10:1596-1598, 1996). Taken together, these studies affirm a route of sexual transmission for KSHV that differs significantly from that for HIV-1.…”

“…Active KSHV replication and increased detection by PCR of viral DNA in the peripheral blood are strongly correlated with (i) increased risk of progression to KS (13,530) and (ii) increased severity of pathogenic stage of KS (47,68); the peripheral viral load predicts the pathogenic outcome of the infection (410). KSHV has also been found in the neutrophil subset of PBMCs (285) (481).…”

Kaposi's Sarcoma-Associated Herpesvirus Glycoprotein K8.1 Is Dispensable for Virus Entry

“…Reagents and enzymes used for TaqMan PCR were obtained from PE Applied Biosystems (Foster City, Calif.). The sequences for the TaqMan FAM probe and ORF37 primers used for the quantitative detection of KSHV molecules were published previously (52) and are listed in Table 1. Each 25-l PCR mixture contained TaqMan universal PCR master mix and 0.25 l of 20 M primer stock for both forward and reverse primers.…”

“…To better quantify the amount of virus produced by transfection of 293 cells, a real-time PCR assay targeting the ORF37 gene was used essentially as described previously (52). The relative amounts of BAC36 DNA and BAC36⌬K8.1 DNA found in supernatants of transfected 293 cells were obtained from a standard curve based on known amounts of BAC36 DNA (see Materials and Methods).…”

Evidence for both Lytic Replication and Tightly Regulated Human Herpesvirus 8 Latency in Circulating Mononuclear Cells, with Virus Loads Frequently below Common Thresholds of Detection

“…A duplex quantitative Taqman PCR system was optimized for HHV-8 orf37 and cellular RNase P. The PCR primers and 6-carboxyfluorescein (FAM)-labeled probe used to quantify orf37 have been described previously (32), but a Black Hole Quencher (BHQ1; Biosearch Technologies, Novato, Calif.) was used instead of 6-carboxytetramethylrhodamine (TAMRA). The forward and reverse primers used for RNase P amplification (kindly provided by Karen McCaustland and Brian Holloway) were 5Ј-AGATTTGGACCTGCGAG CG-3Ј and 5Ј-GAGCGGCTGTCTCCACAAGT-3Ј, respectively; the probe was 5Ј-TTCTGACCTGAAGGCTCTGCGCG-3Ј, labeled at the 5Ј end with 6-carboxy-4,5-dichloro-2,7-dimethoxyfluorescein (JOE) and at the 3Ј end with BHQ.…”