Quantitative analysis of Epstein-Barr virus load by using a real-time PCR assay

Abstract

To measure the virus load in patients with symptomatic Epstein-Barr virus (EBV) infections, we used a real-time PCR assay to quantify the amount of EBV DNA in blood. The real-time PCR assay could detect from 2 to over 10(7) copies of EBV DNA with a wide linear range. We estimated the virus load in peripheral blood mononuclear cells (PBMNC) from patients with symptomatic EBV infections. The mean EBV-DNA copy number in the PBMNC was 10(3.7) copies/microg of DNA in patients with EBV-related lymphoproliferative disorders, 10(4.1) copies/microg of DNA in patients with chronic active EBV infections, and 10(2.2) copies/microg of DNA in patients with infectious mononucleosis. These numbers were significantly larger than those in either posttransplant patients or immunocompetent control patients without EBV-related diseases. In a patient with infectious mononucleosis, the virus load decreased as the symptoms resolved. The copy number of EBV DNA in PBMNC from symptomatic EBV infections was correlated with the EBV-positive cell number determined by the in situ hybridization assay (r = 0.842; P < 0.0001). These results indicate that the real-time PCR assay is useful for diagnosing symptomatic EBV infection and for monitoring the virus load.

FIG. 1

(A) Standard curve for real-time PCR. Serially diluted pGEM-BALF5 plasmid was amplified with or without DNA extraction solutions from blood and analyzed in real time with a model 7700 Sequence Detector. The C_T values were plotted against copy number to construct the standard curve. Explanation of symbols: none, plasmid with EBV insert; PBMNC, PBMNC from an EBV-seronegative patient plus plasmid with EBV insert; plasma, plasma from an EBV negative patient plus plasmid with EBV insert. (B) Effect of heparin on quantitation of plasmid DNA. Serially diluted plasmid controls were amplified with or without various DNA extraction solutions and analyzed using a model 7700 Sequence Detector. Explanation of symbols: none, plasmid with EBV insert; serum, serum from an EBV seronegative patient plus plasmid with EBV insert; serum + EDTA, serum and EDTA plus plasmid with EBV insert; serum + heparin, serum and heparin plus plasmid with EBV insert.

FIG. 2

Quantitation of EBV DNA by real-time PCR. DNA was extracted from PBMNC obtained from patients with symptomatic EBV infections or control patients without EBV-related diseases. Two hundred fifty nanograms of DNA was used for the real-time PCR assay, and the EBV DNA copy numbers per microgram of DNA are shown. Multiple samples for some patients were tested because repeated evaluations were needed. Bars show the means and standard deviations for each group. The dotted line shows the detection limit of the assay.

FIG. 3

Change in the virus load in a patient with IM. Sequential samples from a patient with IM were obtained, and both the PBMNC and plasma were analyzed by the real-time PCR assay. The EBV DNA copy numbers are shown per microgram of DNA. The EBV DNA copy numbers are shown per milliliter of plasma. The dotted line shows the detection limit for each sample.

FIG. 4

Correlation of results of the real-time PCR and in situ hybridization assays. PBMNC were obtained from patients with symptomatic EBV diseases, and 20 samples were analyzed using both the real-time PCR and ISH assays. The copy numbers of EBV-DNA measured by the real-time PCR assay and EBV-positive cell numbers measured by the ISH assay were plotted, and the correlation coefficient (r) was calculated.