Detection of low copy numbers of Epstein-Barr virus by in situ hybridization using nonradioisotopic probes prepared by the polymerase chain reaction.
A highly sensitive in situ hybridization procedure was established using digoxigenin-11-dUTP-labeled probes that were prepared by the polymerase chain reaction (PCR). By using 12 sets of primers, BamHI-W fragment of the Epstein-Barr Virus (EBV) was amplified with labeled substrate in individual PCRs. Then the 12 probes (average size, 120 base pairs) were mixed and hybridized with cultured and RNase-treated Namalwa cells, which contain two copies of EBV genomes per cell. The strength of the signals was much stronger as compared with random-primed probe. Our results indicate that the size-averaged PCR probes magnified the sensitivity for detecting low copy numbers of virus genomes by in situ hybridization and that this technique has the potential for investigating latent virus infection in other clinical situations.
Saeki
K
K
Department of Pathology, Nara Medical University, Kashihara, Japan.
Mishima
K
K
Horiuchi
K
K
Hirota
S
S
Nomura
S
S
Kitamura
Y
Y
Aozasa
K
K
D001483:Base Sequence; D002460:Cell Line; D015342:DNA Probes; D003609:Dactinomycin; D003851:Deoxyribonucleases; D003856:Deoxyuracil Nucleotides; D004076:Digoxigenin; D004854:Herpesvirus 4, Human / Q000302:isolation & purification*; D006801:Humans; D017403:In Situ Hybridization / Q000379:methods*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012260:Ribonucleases; D012680:Sensitivity and Specificity
D015342:DNA Probes; D003856:Deoxyuracil Nucleotides; C027078:deoxyuridine triphosphate; D003609:Dactinomycin; D003851:Deoxyribonucleases; D012260:Ribonucleases; D004076:Digoxigenin