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

Epstein-Barr virus (EBV) is associated with several benign and malignant di seases, and blood tests for EBV viral load show promise as markers of disea se burden in affected patients. A commercial quantitative PCR method (BioSo urce International) was recently introduced to facilitate measuring viral l oad. It relies on coamplification of EBV DNA and a spiked competitor in pla sma or serum, followed by semiautomated product detection on enzyme-linked immunosorbent assay (ELISA) plates. In the current study, analytic performa nce characteristics were assessed, and the authors describe several methodo logic improvements to facilitate laboratory implementation. Rapid DNA extra ction was accomplished using commercial silica spin columns, heat-labile ur acil-N-glycosylase was used to inhibit amplicon contamination, and inexpens ive agarose gels were used to screen for polymerase chain reaction products requiring ELISA plate quantitation. Accuracy and precision were verified u sing EBV DNA standards derived from two cell lines and plasmid containing v iral sequences. The assay was sensitive to as few as five template copies p er 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 y ielded similar viral load results. This commercial EBV viral load assay pro vides sensitive and quantitative detection of EBV DNA using equipment alrea dy available in many molecular diagnostic laboratories.