Monitoring of Epstein-Barr Virus DNA Load in Peripheral Blood by Quantitative Competitive PCR

Abstract

A competitive quantitative PCR (Q-PCR) assay combined with simple silica-based DNA extraction was developed for monitoring of Epstein-Barr virus (EBV) DNA load in unfractionated peripheral blood. The Q-PCR is based on competitive coamplification of a highly conserved 213-bp region of the EBNA-1 open reading frame with an internal standard (IS), added in a known concentration. The IS has the same amplicon length and base composition as the wild-type (WT) EBNA-1 amplicon but differs in 23 internally randomized bases. Competitive coamplification yields two PCR products that are quantified by enzyme immunoassay or by electrochemiluminescence detection, with probes specific for the 23 differing internal nucleotides. The Q-PCR has a sensitivity of 10 copies of either WT or IS plasmid DNA. The Q-PCR was validated by quantification of known amounts of plasmid containing the WT EBNA-1 target. Furthermore, we determined EBV genome copy numbers in different cell lines. For EBV quantification in clinical samples, DNA was isolated from lysed whole blood by silica-affinity purification. Forty-six percent of healthy donor peripheral blood samples were positive by Q-PCR. In most of these samples, viral load was less than 2,000 EBV copies/ml of blood. In peripheral blood samples from two AIDS-related non-Hodgkin’s lymphoma patients, elevated EBV loads (up to 120,000 copies/ml) were observed, which decreased upon therapy. In Burkitt’s lymphoma patients, up to 4,592,000 EBV genome copies/ml of blood were detected. In conclusion, the EBNA-1-based Q-PCR assay provides a reproducible, accurate, and easy method for studying the relationship between EBV load and clinical parameters.

Quantification of Epstein‐Barr virus load in peripheral blood of human immunodeficiency virus‐infected patients using real‐time PCR

“…Using this real-time PCR assay, EBV DNA in PBMCs was detected in 50% of the healthy EBV-seropositive adult individuals, in agreement with previous studies [Gopal et al, 1990;Kimura et al, 1999;Maeda et al, 1999;Stevens et al, 1999;Yamamoto et al, 1995]. The EBV loads ranged from`10 to 485 copies per 10 6 PBMCs in this control group, also in agreement with the low or undetectable levels previously reported in the literature [Wagner et al, 1992;Bai et al, 1997;Rowe et al, 1997;Maeda et al, 1999;Baldanti et al, 2000].…”

Vesicle‐bound EBV‐BART13‐3p miRNA in circulation distinguishes nasopharyngeal from other head and neck cancer and asymptomatic EBV‐infections

“…Next to serology, determining the cell free EBV-DNA load has been extensively evaluated for noninvasive NPC diagnosis. 10,12 However, given that the origin of EBV-DNA in the blood is unclear, diagnosis of primary NPC relying solely on the detection of (fragmented) EBV DNA in circulation may still not be specific enough, or practical, for large scale screening. 15,16,49 This is especially true for poorer To discern what approach would be optimal for early NPC detection we contend that practical and affordable methods need to be compared directly.…”

Epstein–Barr viral load as a marker of lymphoma in AIDS patients

“…Less well studied is the extent to which blood levels of EBV DNA are elevated in AIDS lymphoma patients, whether their tumor is primary to the brain or originating in a peripheral site. Promising data has been reported in small series of AIDS patients in whom EBV load was elevated in serum, in plasma, or in blood mononuclear cells at the time of lymphoma diagnosis, with levels subsequently falling during therapy [Laroche et al, 1995;Stevens et al, 1999;Bossolasco et al, 2002;Van Baarle et al, 2002].…”