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. 2000 Feb;38(2):712-5.
doi: 10.1128/JCM.38.2.712-715.2000.

Development of a real-time quantitative assay for detection of Epstein-Barr virus

H G Niesters  1 , J van Esser, E Fries, K C Wolthers, J Cornelissen, A D Osterhaus
Affiliations

Development of a real-time quantitative assay for detection of Epstein-Barr virus

H G Niesters et al. J Clin Microbiol. 2000 Feb.

Abstract

With the use of real-time PCR, we developed and evaluated a rapid, sensitive, specific, and reproducible method for the detection of Epstein-Barr virus (EBV) DNA in plasma samples. This method allowed us to screen plasma and serum samples over a range between 100 and 10(7) copies of DNA per ml using two sample preparation methods based on absorption. A precision study yielded an average coefficient of variation for both methods of less than 12%, with a coefficient of regression for the standard curve of a minimum of 0. 98. We detected EBV DNA in 19.2% of plasma samples from immunosuppressed solid-organ transplant patients without symptoms of EBV infections with a mean load of 440 copies per ml. EBV DNA could be detected in all transplant patients diagnosed with posttransplant lymphoproliferative disorder, with a mean load of 544,570 copies per ml. No EBV DNA could be detected in healthy individuals in nonimmunosuppressed control groups and a mean of 6,400 copies per ml could be detected in patients with infectious mononucleosis. Further studies revealed that the inhibitory effect of heparinized plasma could be efficiently removed by use of an extraction method with Celite as the absorbent.

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Figures

FIG. 1
FIG. 1
Standard curve for TaqMan PCR. Serial dilutions of the EBV EM standard ranging from 50 to 107 copies per ml were made. Extraction was performed by the method of Boom et al. (1) (triangles; Spearman correlation coefficient, 0.997) or the method with the High Pure Viral Nucleic Acid kit (squares; Spearman correlation coefficient, 0.998) as a matrix. Equal volumes of input and output material (100 μl) were used. The Ct values, which correspond to the PCR cycle number in which the value is above the threshold limit, are plotted against the calculated number of particles counted by electron microscopy.
FIG. 2
FIG. 2
Detection of EBV EM standard dilutions made in heparin-treated plasma (dashed line) or EDTA-treated plasma (solid line). From these EBV dilutions, ranging from 500 to 107 copies per ml, DNA was isolated by the extraction method of Boom et al. (1), which uses Celite (■) to absorb the DNA, as well as by the method with the High Pure Viral Nucleic Acid kit (Roche Diagnostic) (●). The extracted DNA was quantified by the TaqMan assay.
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