Epstein-Barr virus quantitation by real-time PCR targeting multiple gene segments: a novel approach to screen for the virus in paraffin-embedded tissue and plasma.
Epstein-Barr Virus (EBV) infects nearly all humans and then persists for the life of the host. In some people who later develop cancer, EBV DNA is present within malignant cells and circulates at elevated levels in the plasma. In the current study, we validated five novel quantitative polymerase chain reaction (Q-PCR) assays targeting disparate but highly conserved segments of the EBV genome (BamH1W, EBNA1, LMP1, LMP2, and BZLF1). Each assay was sensitive to as few as 50 copies of EBV DNA per reaction and was linear across at least four orders of magnitude. When applied to paraffin-embedded tissues in concert with EBV-encoded RNA (EBER) in situ hybridization, the BamH1W and EBNA1 assays were the most informative, while use of the entire battery of EBV PCR assays may help identify genomic polymorphisms or deletions. Higher viral loads were found in the 17 EBER-positive compared with the 13 EBER-negative tumors (means 84,978 versus 22 copies of EBV per 100,000 cells, respectively). The five Q-PCR assays were also informative in plasma samples where EBV was measurable in all nine patients with lymphoma or infectious mononucleosis, whereas EBV was undetectable in all nine healthy controls. The findings suggest that Q-PCR is an effective method of distinguishing disease-associated virus from incidental virus in paraffin-embedded tissue and in plasma samples.
Ryan
Julie L
JL
Department of Pathology, University of North Carolina at Chapel Hill, NC 27599-7525, USA.
Fan
Hongxin
H
Glaser
Sally L
SL
Schichman
Steven A
SA
Raab-Traub
Nancy
N
Gulley
Margaret L
ML
D045744:Cell Line, Tumor; D004279:DNA, Viral / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000235:genetics; D017353:Gene Deletion; D016679:Genome, Viral; D004854:Herpesvirus 4, Human / Q000235:genetics*; D006801:Humans; D007150:Immunohistochemistry; D017403:In Situ Hybridization; D010232:Paraffin; D016612:Paraffin Embedding; D016133:Polymerase Chain Reaction / Q000379:methods*; D011110:Polymorphism, Genetic; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D015534:Trans-Activators / Q000096:biosynthesis / Q000235:genetics; D014763:Viral Matrix Proteins / Q000096:biosynthesis / Q000235:genetics; D014764:Viral Proteins / Q000096:biosynthesis / Q000235:genetics
C063676:BZLF1 protein, Herpesvirus 4, Human; D004279:DNA, Viral; D004268:DNA-Binding Proteins; C037662:EBV-associated membrane antigen, Epstein-Barr virus; D015534:Trans-Activators; D014763:Viral Matrix Proteins; D014764:Viral Proteins; D010232:Paraffin