title: Performance characteristics of two real-time PCR assays for the quantification of Epstein-Barr virus DNA.
abstract: We compared the performance of a laboratory-developed 5'-nuclease real-time polymerase chain reaction assay and a commercial assay (LightCycler, Roche Diagnostics, Indianapolis, IN) for quantification of Epstein-Barr virus (EBV) DNA. Using standards provided by the manufacturer, the LightCycler assay was linear from 100 to 1 million copies per reaction. Based on dilution of a plasmid containing the amplicon, the laboratory-developed assay was linear from 22 to 45 million copies per reaction. Both assays detected 0.5 copies of genomic EBV DNA per reaction; both showed good reproducibility with coefficients of variation from 0.3% to 2.4% for the LightCycler and 1.8% to 5.1% for the laboratory-developed assay. For 31 whole blood specimens with measurable values in both assays, the viral load values obtained with the LightCycler averaged 2.3-fold higher than those obtained with the laboratory-developed assay. Testing 30 matched whole blood and plasma samples in the laboratory-developed assay showed whole blood viral load values averaged 10-fold higher than those for plasma. The LightCycler and laboratory-developed assays are sensitive and reproducible with broad linear ranges. Further clinical evaluation is needed to determine the viral load cutoff that is predictive of posttransplantation lymphoproliferative disorders.
authors.affiliation: Department of Pathology and Laboratory Medicine, Emory University School of Medicine, 1364 Clifton Road, Atlanta, GA 30322, USA.
authors.forename: Charles E
authors.initials: CE
authors.lastname: Hill
authors.forename: Shealynn B
authors.initials: SB
authors.lastname: Harris
authors.forename: Elizabeth E
authors.initials: EE
authors.lastname: Culler
authors.forename: James C
authors.initials: JC
authors.lastname: Zimring
authors.forename: Frederick S
authors.initials: FS
authors.lastname: Nolte
authors.forename: Angela M
authors.initials: AM
authors.lastname: Caliendo
mesh_terms: D004279:DNA, Viral / Q000097:blood*; D020031:Epstein-Barr Virus Infections / Q000175:diagnosis* / Q000821:virology; D004854:Herpesvirus 4, Human / Q000235:genetics* / Q000254:growth & development / Q000302:isolation & purification; D008232:Lymphoproliferative Disorders / Q000175:diagnosis / Q000209:etiology / Q000821:virology; D016377:Organ Transplantation / Q000009:adverse effects; D010949:Plasma; D011183:Postoperative Complications; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D019562:Viral Load
chemical_list: D004279:DNA, Viral