Hx. Fan et al., Analytic validation of a competitive polymerase chain reaction assay for measuring Epstein-Barr viral load, DIAGN MOL P, 10(4), 2001, pp. 255-264
Citations number
25
Categorie Soggetti
Research/Laboratory Medicine & Medical Tecnology","Medical Research Diagnosis & Treatment
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.