Oligonucleotides for polymerase chain reaction amplification and hybridization detection of Epstein-Barr virus DNA in clinical specimens.
We designed synthetic oligonucleotide primers and hybridization probe for use in polymerase chain reaction (PCR) amplification and hybridization detection of Epstein-Barr virus (EBV) nucleic acid sequences. Primer sequences were chosen from the coding region for the Epstein-Barr virus nuclear antigen-1 (EBNA-1). PCR amplification and hybridization with these oligonucleotides was carried out on standard laboratory cell lines including African Burkitt's lymphoma and infectious mononucleosis derived cell lines, as well as cell lines recently established from clinical EBV isolates from bone marrow transplant recipients. All EBV cell lines tested were positive. No false-positives were detected with uninfected cell lines, human placental DNA or with other viruses. The sensitivity of the detection procedure was such that four copies of the EBV genome could consistently be detected in a background of 1 microgram of placental DNA. EBV was detected in DNA extracts from the peripheral blood mononuclear cells of two patients with infectious mononucleosis and one patient with viral-associated hemophagocytic syndrome. Three of 18 EBV seropositive patients without known ongoing EBV-associated illness undergoing ambulatory surgery also had EBV detected in DNA extracts from their peripheral blood mononuclear cells. EBV was detected in DNA extracts from lymphoma tissue from two patients with post-transplant lymphomas and two AIDS patients with primary CNS lymphomas. EBV was not detected in 12 B-cell lymphoma specimens from patients without history of immunocompromise. DNA extracts from formalin-fixed paraffin-embedded Hodgkin's tissues previously shown to be EBV positive by Southern blot were also demonstrated to be EBV positive by PCR. Thus, with the oligonucleotides described, PCR is applicable to the detection of EBV in a spectrum of clinical isolates. The broad specificity of these oligonucleotides for all strains of EBV tested is probably a function of the highly conserved sequence of the EBNA-1 DNA binding domain.
Ambinder
R F
RF
Johns Hopkins Oncology Center, Baltimore, MD 21231.
Lambe
B C
BC
Mann
R B
RB
Hayward
S D
SD
Zehnbauer
B A
BA
Burns
W S
WS
Charache
P
P
D000956:Antigens, Viral / Q000235:genetics; D015139:Blotting, Southern; D002051:Burkitt Lymphoma / Q000473:pathology; D004273:DNA, Neoplasm / Q000032:analysis; D004279:DNA, Viral / Q000235:genetics / Q000302:isolation & purification*; D019309:Epstein-Barr Virus Nuclear Antigens; D006564:Herpesviridae / Q000235:genetics; D004854:Herpesvirus 4, Human / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D007244:Infectious Mononucleosis / Q000382:microbiology; D008223:Lymphoma / Q000382:microbiology; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction; D011237:Predictive Value of Tests; D014407:Tumor Cells, Cultured / Q000382:microbiology; D014412:Tumor Virus Infections / Q000175:diagnosis* / Q000382:microbiology
D000956:Antigens, Viral; D004273:DNA, Neoplasm; D004279:DNA, Viral; D019309:Epstein-Barr Virus Nuclear Antigens; D015345:Oligonucleotide Probes