Quantitative Analysis of Epstein-Barr Virus Load by Using a Real-Time PCR Assay

Abstract

To measure the virus load in patients with symptomatic Epstein-Barr virus (EBV) infections, we used a real-time PCR assay to quantify the amount of EBV DNA in blood. The real-time PCR assay could detect from 2 to over 107 copies of EBV DNA with a wide linear range. We estimated the virus load in peripheral blood mononuclear cells (PBMNC) from patients with symptomatic EBV infections. The mean EBV-DNA copy number in the PBMNC was 103.7 copies/μg of DNA in patients with EBV-related lymphoproliferative disorders, 104.1 copies/μg of DNA in patients with chronic active EBV infections, and 102.2copies/μg of DNA in patients with infectious mononucleosis. These numbers were significantly larger than those in either posttransplant patients or immunocompetent control patients without EBV-related diseases. In a patient with infectious mononucleosis, the virus load decreased as the symptoms resolved. The copy number of EBV DNA in PBMNC from symptomatic EBV infections was correlated with the EBV-positive cell number determined by the in situ hybridization assay (r = 0.842; P < 0.0001). These results indicate that the real-time PCR assay is useful for diagnosing symptomatic EBV infection and for monitoring the virus load.

Recent advancements for the evaluation of anti-viral activities of natural products

“…Mathematical analysis of the data, and comparison to control reactions containing known amounts of template, allows one to calculate the amount of input DNA in the initial reaction. The severity of some diseases has been shown to correlate with the viral load, making real-time PCR quantitation useful to study, not simply the presence of a virus but the role of viral reactivation or persistence in the progression of the disease [27][28][29]. This property has been successfully adapted to screen potential anti-viral test compounds in vitro.…”

Detection and monitoring of virus infections by real-time PCR

“…Since the fluorescence generated during the amplification reaction is proportional to the amount of PCR product, semi-quantitative estimation of virus copies in the specimen investigated is feasible even without precise calibration of the assay, which is required for truly quantitative analysis (see Section 5.1). A large number of real-time PCR assays for the detection of viral pathogens, including both DNA and RNA viruses, have been described (Abe et al, 1999;Kimura et al, 1999;Ryncarz et al, 1999;Kleiber et al, 2000;Lallemand et al, 2000;Monpoeho et al, 2000;Tanaka et al, 2000;Ohyashiki et al, 2000;Furuta et al, 2001;Watzinger et al, 2004). The latter require special care in specimen processing because of the susceptibility of RNA to the digestion by ribonucleases that may be present in clinical samples.…”

Real-time Fluorescent PCR Techniques to Study Microbial–Host Interactions

“…Techniques to Study acid template is a significant benefit for microbial load determination (Ishiguro et al, 1995;Kimura et al, 1999;Najioullah et al, 2001;Ryncarz et al, 1999;Monopoeho et al, 2000;Alexandersen et al, 2001;Abe et al, 1999;Locatelli et al, 2000;Gruber et al, 2001;Brechtbuehl et al, 2001;Moody et al, 2000). This broad dynamic range obviates the need to dilute an amplicon before detecting it or the repetition of an assay using a diluted template because a preliminary result falls above the upper limits of the detection assay.…”

“…Quantitative real-time PCR has similarly examined the interaction between virus and host and monitored changes in viral load resulting from antiviral therapy ultimately impacting on the treatment regimen selected (Nitsche et al, 1999;Clementi, 2000;Limaye et al, 2000). Because disease severity and viral load are linked, microbial quantitation by real-time PCR has proven beneficial when studying the impact of viral reactivation, persistence or isolated gene expression on the progression of disease (Ohyashiki et al, 2000;Chen et al, 2003;Kearns et al, 2001c;Furuta et al, 2001;Limaye et al, 2001;Tanaka et al, 2000a,b;Lallemand et al, Real-time Fluorescent PCR Techniques to Study 2000;Laue et al, 1999;Kimura et al, 1999;Chang et al, 1999;Lo et al, 1999;Hawrami and Breur, 1999;Hoshino et al, 2000;Nitsche et al, 2000;Machida et al, 2000;Limaye et al, 2001;Najioullah et al, 2001;Gault et al, 2001). Altered microbial tropism or replication and the effect of these changes on the host cell can also be followed using real-time PCR (Kennedy et al, 1998a(Kennedy et al, , 1999a.…”