TABLE 19.

Quantitative detection of Epstein-Barr virus DNA by real-time PCRa

PlatformAuthor (reference)SpecimenTargetQuantitative standardReporting unitsComparison to other systemsComments
ABI PrismKimura et al. 1999 (221)Peripheral blood mononuclear cells (PBMC) BALF5 gene (DNA polymerase)Linear range, 2 to 107 copies EBV DNA/μg DNACopies EBV DNA/μg of PBMC DNAReal-time PCR detection of EBV DNA was compared with in situ hybridization techniques.The virus load in peripheral mononuclear cells was 103.7 copies/μg of DNA in patients with EBV-related lymphoproliferative disorders, 104.1 copies/μg DNA in patients with chronic active EBV infections, and 102.3 copies/μg DNA in patients with infectious mononucleosis. The copy numbers of EBV DNA in PBMC from symptomatic EBV infections was correlated with the EBV-positive cell number determined by the in situ hybridization assay (P < 0.0001).
Lo et al. 1999 (281; also 265, 280)PlasmaBamHI-W; EBNA-1A calibration curve was run in parallel and in duplicate with each analysis, using DNA extracted from the EBV-positive cell line Namalwa (ATCC) as a standard. A conversion factor of 6.6 pg of DNA/diploid cell was used for copy number calculation.Copies EBV DNA/mlUsing real-time PCR cell-free EBV DNA was detectable in 55/57 (96%) median concentration, 21,058 copies of nasopharyngeal carcinoma (NPC) patients and 3/43 (7%) controls (median concentration, 0 copies/ml). Results suggest quantitative analysis of plasma EBV DNA may be a useful clinical and research tool in the screening and monitoring of patients with nasopharyngeal carcinoma.
Niesters et al. 2000 (342)Plasma BNRF1 p143 gene (nonglycosylated membrane protein)A standard containing 6.68 × 109 EBV particles/ml (Advanced Biotechnologies) was used as a standard. Serial half-log dilutions of this standard ranging from 107 to 10 copies/ml were made to characterize linearity, precision, specificity, and sensitivity of the assay.Copies EBV DNA (genome equivalents)/mlEBV DNA could be detected in all transplant patients diagnosed with PTLD, with a mean load of 544,570 copies/ml. No EBV DNA could be detected in healthy individuals and in immunosuppressed control groups. A mean of 6,400 copies/ml could be detected in patients with infectious mononucleosis.
Orii et al. 2000 (356)22 transplant patients BALF5 A plasmid that contained BALF5 was constructed and diluted to prepare a standard source curve for quantitation.Copies EBV/μg DNAQuantitative real-time PCR was compared to qualitative DNA in plasma by PCR and EBV-encoded mRNAReal-Time PCR and EBER-1 (in situ hybridization) results exceeded the cut-off level of 102.5 copies/μg DNA
(EBER1) by in situ hybridization for the detection of posttransplant lymphoproliferative disease
Dehee et al. 2001 (91)Blood, peripheral blood mononuclear cells BNRF1 geneA plasmid with the target gene was developed to produce a standard curve for quantitative determinations. All virus DNA quantification was carried out simultaneously in order to determine the input cellular DNA for each sample and was used as an endogenous reference to normalize the variations due to differences in the PBMC count or DNA extraction. The Namalwa cell line (ATCC CRL1432) containing two integrated copies of the EBV genome/cell was used as a positive control.Copies EBV DNA/106 PBMCSignificantly higher EBV loads were found in HIV-infected patients compared with EBV-seropositive healthy group (P < 0.00001). EBV loads were not correlated with the clinical stages of HIV infection or HIV replication
Jabs et al. 2001 (198)Peripheral blood mononuclear cellsBamHI-K (BKRF1) encoding EBNA-1A pCMV EBNA plasmid which carries the complete EBNA nuclear gene (EBNA-1) was used for calibration of the Namalwa DNA standard (CRL-1432, ATCC)Copies of EBV DNA/μg of PBMC DNAAim of study was to develop a rapid and reliable PCR protocol for the quantification of cell-associated EBV DNA associated with PTLD in transplant recipients.Single-tube coamplification of EBV and genomic coreactive proteins allowed normalization of EBV DNA copy levels that provided a more accurate quantification (than amplification of EBV DNA alone) of cell bound target DNA.
Lo et al. 2001 (282)SerumBamHI-WA calibration curve was run in parallel and in duplicate with each analysis, using DNA extracted from the EBV-positive cell line Namalwa as a standard. A conversion factor of 6.6 pg of DNA/diploid cell was used for copy number calculation (281).Copies EBV DNA/mlPatients with gastric carcinoma, gastritis, and healthy controls were evaluated for the presence of EBV DNA in serum by real-time PCR. Resected tumor specimens from patients with gastric carcinoma were tested for EBER (in situ) and by real-time PCR (serum)Serum EBV DNA reflects tumoral EBER status and presents the possibility that circulating EBV DNA may be used as a tumor marker for the EBER-positive gastric carcinomas.
van Esser et al. 2001 (509)Plasma (stem cell transplant patients) BNRF1 p143 nonglycosylated membrane protein geneA standard containing 6.68× 109 EBV particles/ml (Advanced Biotechnologies) was used as a standard. Serial half-log dilutions of this standard, ranging fromCopies EBV DNA (genome equivalents)/mlQualitative monitoring of EBV DNA levels from the start of and during therapy for EBV-lymphoproliferative disease rapidly and
107 to 10 copies/ml were made to characterize linearity, precision, specificity, and sensitivity of the assay.accurately predicts for response to therapy as early as within 72 h.
Wagner et al. 2001 (525)Blood, peripheral blood mononuclear cells plasmaBamHI-K region coding for EBNA1 BamHI-WThe EBV-positive Burkett's lymphoma cell line Namalwa was used as a standard for quantification of EBV DNA. Namalwa cells contain two integrated EBV copies/cellular genome. Other cell lines (Daudi and Raji) were also used as positive controls.EBV genomes/μg PBMC DNAPatients with PTLD had a median viral load of 19,200 EBV genomes/μg PBMC DNA or 3,225 EBV genomes/100μ l plasma. Although both PBMC and plasma were useful specimens for laboratory diagnosis of PTLD, the specificity was higher if the EBV viral load was determined in plasma.
Leung et al. 2002 (264)Whole blood BALF5 gene (DNA polymerase)Raji cells used as a positive control for detection and preparation of a standard curve for EBV DNA (dilutions to 7.5 105 genome/reaction; β-globulin used as a housekeeping gene.Copies EBV DNA/μg amplified DNA (EBV DNA copy number was normalized againstβ -globulin)Aim was to develop a rapid and reliable PCR protocol for quantitation of the cell-associated EBV genome.The mean copy levels of EBV DNA/μg amplified DNA in patient populations were: healthy controls, 0.5; hemodialysis, 27; renal transplant, 40; and infectious mononucleosis, 782. The percentage of individuals whose EBV levels were >35 copies/μg amplified DNA: healthy, 0; hemodialysis, 38; renal transplant, 40%; infectious mononucleosis, 87%.
Matsukura et al. 2002 (305)Blood (PBMC) BALF5 gene (DNA polymerase)A standard curve for quantitative detection of EBV DNA was obtained by measuring dilutions of a plasmid containing the target gene.Copies EBV DNA/μg DNAAim of study was to demonstrate the significance of serial monitoring of EBV DNA by real-time PCR after liver transplantation.In 15 patients, the mean values of the highest EBV DNA levels from patients who had the following clinical features: fever (36,232); upper respiratory syndrome (16,040); diarrhea (15,968); ascites (2,485); lymphadenopathy (336,858); and PTLD (60,486).
Teramura et al. 2002 (487)Plasma; serum BALF5 gene (DNA polymerase)A standard curve for quantitative detection of EBV DNA was obtained by measuring dilutions of a plasmid containing the target gene.Copies EBV DNA/mlStudy compared the detection of EBV DNA in specimens from patients with hemophagolytic lymphohistiocytosis with patients with infectious mononucleosis.EBV DNA copy levels from serial specimens from 10 patients demonstrated decreasing or undetectable levels of target DNA following appropriate therapy.
van Esser et al. 2002 (510)Plasma (stem cell transplant patients) BNRF1 p143 gene (nonglycosylated membrane protein)A standard containing 6.68 × 109 EBV particles/ml (Advanced Biotechnologies) was used as a standard. Serial half-log dilutions of this standard, ranging from 107 to 10 copies/ml were made to characterize linearity, precision, specificity, and sensitivity of the assay.Copies EBV DNA (genome equivalents)/mlAuthors studied whether preemptive therapy with rituximab prevents EBV lymphoproliferatifive disease in patients undergoing stem cell transplantation.Preemptive therapy was given to patients with viral reactivation more than or equal to 1,000 copies of EBV DNA (genome equivalents)/ml; 14/17 (82.4%) patients treated with rituximab had complete clearance of EBV DNA from plasma.accurate quantification (than amplification of EBV DNA alone) of cell bound target DNA.
Orentas et al. 2003 (355)Blood, peripheral blood mononuclear cells EBER1 geneA plasmid containing the EBER1 gene was used to prepare a standard curve for the quantitative detection of EBV DNA.EBV genomes/μg PBMCIn 9 patients (8 bone marrow, 1 kidney transplant), PTLD was associated with a rapid rise in viral load exceeding 105 EBV genomes/μg of PBMC-derived DNA. The threshold for normal EBV viral load (compared to the levels in patients with PTLD) based on a combined experience with viral load analysis is defined as 104 EBV genomes/μg PMBC.
Pitetti et al. 2003 (381)Serum, Patient population was children (mean age, 9 yr) with primary EBV infection (n= 28); EBV seronegative patients (n= 25); EBV-seropositive patients (n= 26) BALF5 gene (DNA polymerase)Plasmid with target insert was used to provide quantitation standards.Copies EBV DNA/mlNone of the purified DNA sample inhibited the amplification of the internal positive control. Twenty-one (75%) of the patients with primary EBV, one (4%) of the seronegatives, and none of the seropositives had detectable EBV DNA loads. Viral loads varied widely in patients with primary EBV infection (101 to 103). EBV posttransplant lymphoproliferative disease was diagnosed in a seronegative patient with an EBV DNA load of 1,450 copies/ml.
Wadowsky et al. 2003 (524)Whole blood; plasma from 44 transplant patients BALF5 gene (DNA polymerase)The gene target was inserted into a plasmid and determinations made in 10-fold increments rangingPlasma: copies EBV DNA/mlWhole blood: EBV DNA copies/ml, EBV copies/μg DNA based on spectrophotometricComparing real-time PCR and conventional PCR, whole-blood PBL loads correlated highly (r2=
from 20 to 2,000,000 copies/reactionmeasurement, and EBV copies DNA/105 PBLs based on absolute count. EBV DNA load levels were measured in whole blood (n = 60), plasma (n= 59), and samples of peripheral blood lymphocytes (n = 6) by competitive PCR.0.900), whereas plasma and PBL loads correlated poorly (r2 = 0.512). Data provide outoff levels of EBV DNA in blood compartments (whole blood, plasma, PBLs) for various risk groups of patients.
Leung et al. 2004 (266)PlasmaBamHI-W EBNA-1A calibration curve was run in parallel and in duplicate with each analysis, using DNA extracted from the EBV-positive cell line Namalwa as a standard. A conversion factor of 6.6 pg of DNA/diploid cell was used for copy number calculation (281).Copies EBV DNA/mlThe sensitivities and specificities of IgA-VCA and EBV DNA for diagnosis of NPC were determined in 139 new cases of NPC and 178 healthy individuals.The sensitivities of EBV DNA and IgA-VCA for diagnosis of NPC were 95% and 81%, respectively. The specificities of EBV DNA and IgA-VCA were 98% and 96%, respectively.
Lin et al. 2004 (273)Plasma specimens from 99 patients with biopsy-proven nasopharyngeal carcinomaABI Prism/BamHI-Wβ-Globulin gene served as a control for activity of Taq polymerase. A calibration curve was obtained using DNA extracted from the EBV-positive cell line Namalwa as the standard.EBV DNA/ml plasmaThe median concentrations of plasma EBV DNA were 681 copies/ml among 25 patients with stage III disease, 1,703 copies/ml among 74 patients with stage IV disease, and 291,940 copies/ml among 19 control patients with distant metastasis. Patients with relapse had a significantly higher plasma EBV DNA concentration before treatment than those who did not have a relapse. Quantification of plasma EBV DNA was useful for monitoring patients with nasopharyngeal carcinoma and predicting the outcome of treatment.
Yu et al. 2004 (555)PlasmaEBERAll plasma DNA samples were also subjected to real-time PCR analysis for the β-globin gene.Copies EBV DNA/mlTumor tissue samples were tested for the presence of EBV by in situ hybridization and compared to the detection of EBV DNA in plasma by real-time PCR.Plasma EBV DNA concentrations in patients with EBV-encoded RNA (EBER)-positive tumors nonnasopharyngeal and neck carcinomas (squamous cell carcinoma; lymphoepithelial carcinomas) (median, 3,827 copies) were significantly
    higher than those in the controls (median, 0 copy/ml, P = 0.0001). Plasma EBV DNA was detected in all of the patients with EBER-positive tumors.
EBV LightCyderAritaki et al. (21)Peripheral blood cells; 16 patients undergoing stem cell transplantation.GenBank accession no. V01555Raji cells that contained 50 copies EBV genome/cell used as standardCopies/μg of EBV DNAOther herpesvirus targets (HHV-6 and CMV) were measured in this study.No EBV-related disease was found in the study patients. Viral loads were low (<103 copies/μg of EBV DNA). High numbers of CMV genome were detected in 3/13 patients after transplant and reactivation of HHV-6 was frequently seen.
Brengel-Pesce et al. (45)Peripheral blood mononuclear cells from 88 patients: 32 healthy EBV-seropositive; 34 EBV-associated disease; 22 HIV infected. BXLF-1 gene (thymidine kinase) BXLF-1 cloned into plasmid and quantitative standard run from 2 to 2 × 107 copies.PBMC: copies EBV DNA genome/μg DNA. Serum: copies EBV DNA/ml.LightCycler results were compared with a routinely used ELISA-PCR of 150 samples and a good correlation was found (R = 0.956)12/32 (37.5%) PBMC from healthy EBV-seropositive individuals were positive at very low copy levels. EBV DNA was detected in 80% of classical infection mononucleosis patients (mean, 288 copies/ml of serum). In 5 cases of EBV-related posttrans plantation lymphoproliferative disease, the viral load was >10,000 copies EBV DNA/μg DNA at the diagnosis of lymphoma.
Stevens et al. (463)Whole blood EBNA-1 geneEBNA was inserted into plasmid DNA and quantified in duplicate in each PCR run. For a quantitative standard the plasmid was used in concentrations ranging from 10 to 104 copies/reaction.Copies EBV DNA/mlReal-time PCR was compared with quantitative competitive PCR for EBV DNA.Aim of study was to develop a LightCycler-based real-time PCR assay for monitoring EBV load in whole blood. In 253 blood samples from patients with Burkitt's lymphoma, infectious mononucleosis, or human immunodeficiency virus infection, a weak but significant correlation between the two methods was found (P < 0.001).
Patel et al. (370)Whole blood; plasma BZLF-1 gene, which codes for the ZEBRA proteinNamalwa EBV cell line (ATCC 1432) used as a standard, which contains two copies EBV genome/cell. BZLF-1 gene was incorporated into a plasmid for use as a quantitative control (100 to 109 copies/reaction).Copies CMV DNA/mlIntra- and interassay variability studies using external quantitative standards and DNA extracted from whole blood and plasma samples were shown to be within 0.5 log10.Quantitative EBV DNA assay was developed to investigate the natural history of EBV infection in immunosuppressed patients to identify those at greatest risk of developing disorders and monitor response to therapy.
Stöcher et al. (466, 467)PlasmaGenBank accession numbers 109336-109351 (EBV). Note: numbers for CMV, HSV-1/2 and VZV also included.Copies EBV DNA/mlInternal control DNA contained a stretch of the neomycin phosphotransferase gene which was flanked by the four forward and reverse primer binding sites that were specific for each herpesvirus type-specific PCR of the set.Authors developed a set of automated LightCycler PCR assays for the detection of CMV, EBV, HSV-1/2, and VZV DNA in plasma samples and complementation of the assays with internal amplification controls.
LightCyclerBalandraud et al. (24)Peripheral blood mononuclear cellsIR-1Raji cell line, which harbors 50 copies of EBV genome per cell, was used as an external EBV standard. EBV copy number was calibrated by serial dilutions of Raji cell DNA and ranged from 105 to 0.064 copies/ml of standard EBV DNA dilution.Copies EBV/500 ng DNA (1.5 × 105 cells)In patients with rheumatoid arthritis, the EBV DNA load in PBMCs is increased almost 10-fold compared with that in normal controls.
iCycler iQYuge et al. (556)Blood, PBMC, and plasma BALF5 gene (DNA polymerase)Linear range, 2 to 107 copies EBV DNA/μg DNA.Copies EBV DNA/μg DNACase report of a 3-year-old, previously healthy boy who developed a chronic active EBV infection.Viral loads of EBV DNA in PBMC and plasma remained high with therapeutic interventions. PCR of a liver tissue sample was positive for EBV DNA CD56+ cells infected with EBV.
Jeblink et al. (200)Whole blood EBER gene (glycoprotein B gene for CMV)Both EBV and CMV assays were able to detect viral DNA over a linear range of 101 to 107 copies/well, which was equivalent to 103 to 109 copies/mlEBV DNA copies/mlEBV and CMV viral loads in patient samples obtained by gel-based and by real-time PCR were very similar.The real-time PCR assays showed increases in viral load before clinical measures of viral disease and decreases in viral load during antiviral therapy in two of six pediatric patients.
  • a Abbreviations: ATCC, American Type Culture Collection; PTLD, posttransplant lymphoproliferative disease; HIV, human immunodeficiency virus; PBL, peripheral blood leukocytes; IgA, immunoglobulin A; VCA, viral capsid antigen.