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.

FIG. 1

Cloning of the IS. Primers C-EBNA1-1 and C-EBNA1-2 were designed with 23 randomized bases at the 5′ end. Two separate PCRs with primers C-EBNA1-2 and QP1 and primers C-EBNA1-1 and QP2 were performed. Purified PCR products were mixed and reamplified with primers QP1 and QP2. A BamHI K rightward frame 1 (BKRF1) PCR product with 23 internally randomized bases compared to the wild-type gene was obtained and cloned into pCR-Script SK(+), giving pQPCR-8.

FIG. 2

Analytical sensitivity of EBNA-1 Q-PCR assay for WT and IS plasmid DNA targets. WT or IS plasmid DNA was quantified by spectrophotometry, and dilution series ranging from 10⁵ to 10⁻¹ copies of the plasmid were amplified in EBNA-1 PCR. PCR products were detected by Southern blotting and hybridization with either WT- or IS-specific radiolabeled oligoprobe. Ten copies of both WT and IS target could be detected.

FIG. 3

Reconstruction series showing equal amplification of WT and IS. Serial 10-fold dilutions of WT were spiked with increasing amounts of IS in separate reactions. WT and IS were competitively coamplified in Q-PCR, and PCR products were detected by Southern blotting and hybridization with a specific radiolabeled oligonucleotide probe (a) or by EIA (b).

FIG. 4

Detection of amplified IS dilution series by ECL.

FIG. 5

Quantification of WT plasmid DNA. One thousand copies of WT plasmid were spiked with 10⁵, 10⁴, 10³, and 10² copies of IS in four separate reactions and amplified in EBNA-1 PCR. PCR products were quantified by EIA, and the logarithm of WT signal/IS signal was plotted against the logarithm of the IS amount.

FIG. 6

EBV load dynamics in peripheral blood of two patients with ARNHL. Patient A (left graph) was diagnosed for ARNHL in February 1995 and treated three times with CHOP therapy. This patient died in June 1995. Patient B (right graph) was diagnosed for ARNHL in January 1995 and treated three times with CHOP therapy and adriamycin-bleomycin-vincristine therapy. In December 1995, this patient was treated with foscavir for cytomegalovirus-associated retinitis. Patient B died in April 1996 due to Kaposi’s sarcoma. D, diagnosis of ARNHL.

FIG. 7

Distribution of EBV loads in peripheral blood of Malawian Burkitt’s lymphoma (BL) patients and controls. Peripheral blood samples of patients were obtained at diagnosis. The control population consisted of patients’ relatives, mostly their mothers.