A comparison of quantitative-competitive and realtime PCR assays using an identical target sequence to detect Epstein-Barr virus viral load in the peripheral blood.
Monitoring the load of Epstein-Barr virus (EBV) in the peripheral blood by quantitative PCR has been accepted as a useful tool for predicting the onset of EBV related diseases, confirming an EBV disease diagnosis and following the response to treatment interventions. In the present study, the use of a realtime polymerase chain reaction (rt-PCR) assay developed for unpurified cell preparations was examined and the results of the realtime assay were compared to an EBV quantitative-competitive PCR assay (QC-PCR). Both assays use the same target sequence and the same method for determining the standard value for the copy number of EBV genomes present. A comparison of 572 PCR results reveals that the realtime assay gave 5-10-fold higher values than the QC-PCR. Fifty-one results (8.9%) were discordant between the two sets of data. The most commonly encountered discordant result was detection of low amounts of EBV DNA by the rt-PCR assay that were not detected in specimens by QC-PCR. The two assays had a high degree of correlation across the range of load detection allowing clinically relevant threshold values determined in the QC-PCR assay to be inferred for the rt-PCR assay. External normalization of the rt-PCR assay was determined to be an important tool for monitoring the quality and/or quantity of human DNA in the starting material. rt-PCR assays with unpurified cell lysates compare favorably with quantitative-competitive assays and when normalized offer real advantages in specimen preparation, assay manipulations and reproducibility over both quantitative-competitive assays and realtime assays that require purified nucleic acid inputs.
Xu
Shushen
S
Department of Infectious Diseases and Microbiology, Graduate School of Public Health, University of Pittsburgh, 130 DeSoto Street, Pittsburgh, Pennsylvania, United States.
Green
Michael
M
Kingsley
Laurence
L
Webber
Steven
S
Rowe
David
D
D001769:Blood / Q000821:virology*; D004279:DNA, Viral / Q000032:analysis*; D004854:Herpesvirus 4, Human / Q000302:isolation & purification*; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013223:Statistics as Topic; D019562:Viral Load
D004279:DNA, Viral