Analytic validation of a competitive polymerase chain reaction assay for measuring Epstein-Barr viral load

Abstract

Epstein-Barr virus (EBV) is associated with several benign and malignant diseases, and blood tests for EBV viral load show promise as markers of disease burden in affected patients. A commercial quantitative PCR method (BioSource International) was recently introduced to facilitate measuring viral load. It relies on coamplification of EBV DNA and a spiked competitor in plasma or serum, followed by semiautomated product detection on enzyme-linked immunosorbent assay (ELISA) plates. In the current study, analytic performance characteristics were assessed, and the authors describe several methodologic improvements to facilitate laboratory implementation. Rapid DNA extraction was accomplished using commercial silica spin columns, heat-labile uracil-N-glycosylase was used to inhibit amplicon contamination, and inexpensive agarose gels were used to screen for polymerase chain reaction products requiring ELISA plate quantitation. Accuracy and precision were verified using EBV DNA standards derived from two cell lines and plasmid containing viral sequences. The assay was sensitive to as few as five template copies per polymerase chain reaction and was linear across four orders of magnitude (correlation coefficient 0.995). When applied to matched plasma and serum samples from 15 patients with nasopharyngeal carcinoma, both sample types yielded similar viral load results. This commercial EBV viral load assay provides sensitive and quantitative detection of EBV DNA using equipment already available in many molecular diagnostic laboratories.