Validation of Roche LightCycler Epstein-Barr virus quantification reagents in a clinical laboratory setting

Abstract

Epstein-Barr virus (EBV) is associated with a wide range of benign and malignant diseases, including infectious mononucleosis, lymphoma, posttransplant lymphoproliferative disorder, and nasopharyngeal carcinoma. Measurement of EBV viral load in plasma is increasingly used for rapid assessment of disease status. We evaluated the performance characteristics of an EBV polymerase chain reaction assay that uses commercial reagents and instruments from Roche Diagnostics (Indianapolis, IN). DNA was extracted from plasma using a MagNaPure instrument, and viral load was measured by real-time polymerase chain reaction on a LightCycler. Analyte-specific reagents included primers and hybridization probes targeting the EBV LMP2 gene and a spiked control sequence. Accuracy and reproducibility were established using DNA from three cell lines. The assay was sensitive to approximately 750 copies of EBV DNA per milliliter of plasma and was linear across at least four orders of magnitude. The assay detected EBV DNA in three of five samples from nasopharyngeal carcinoma patients, seven of nine infectious mononucleosis samples, and 34/34 samples from immunosuppressed patients with clinically significant EBV-related disease, whereas EBV DNA was undetectable in plasma from 21 individuals without EBV-related disease. In conclusion, this LightCycler EBV assay is rapid, sensitive, and linear for quantifying EBV viral load. The assay appears to be useful for measuring clinically significant EBV levels in immunodeficient patients.

Figure 1

EBV assay 1 performed on the Roche Lightcycler is sensitive, accurate, and linear across a 5-log range. Genomic DNA from the Namalwa Burkitt lymphoma cell line was used as template in PCR (A) or was spiked into plasma and then extracted on the MagNaPure instrument before using as template in PCR (B). The amount of input EBV DNA is shown on the x axis, and the amount measured by the assay is shown on the y axis.

Figure 2

EBV levels are shown as measured by three different viral load assays in 48 plasma samples from patients with EBV-related disease. EBV DNA was undetectable by LightCycler assay 1 in six patients who had low-level EBV by the other two assays. In the remaining 42 samples, Lightcycler assay 1 levels tended to be lower than those of the other two assays at levels below 1000 copies per ml, whereas the values were quite similar across the three assays for samples above 1000 copies per ml. It is not clear why the values of all three assays correlated most closely at higher rather than lower viral loads.

Figure 3

Melt-curve analysis reveals an abnormal melt temperature (Tm) in duplicate EBV PCR assays on sample 44. This plasma sample was taken from a woman with terminal multiorgan failure following a drug overdose. A typical melt curve on the right has probe dissociation at approximately 61°C, whereas the atypical melt curves on this particular sample have probe dissociation at approximately 54°C.