Demonstration of Epstein-Barr virus genomes, using polymerase chain reaction in situ hybridization in paraffin-embedded lymphoid tissues.
We used the polymerase chain reaction (PCR) in situ hybridization (ISH) (PCR-ISH) on sections of malignant lymphoma and nonspecific lymphadenitis to detect small amounts of Epstein-Barr virus (EBV), a DNA virus of the herpes virus family. We first surveyed the EBV DNA by Southern blot analysis and PCR, and then compared results of the two PCR/ISH methodologies with the results of simplified/sensitive ISH for the positive cases. The target of the simplified in situ (DNA-ISH) was a few copies of EBV DNA per cell, and the target of the sensitive in situ (RNA-ISH) was as many as 10(7) copies of EBV RNA per cell. When EBV DNA was detected by Southern blot, DNA-ISH, RNA-ISH and PCR-ISH all revealed EBV genomes. When PCR revealed only amplified EBV DNA, DNA-ISH showed no EBV genomes, but PCR-ISH and RNA-ISH showed EBV genomes in a few cells. When PCR showed no detectable amplified EBV DNA, all of DNA-ISH, RNA-ISH and PCR-ISH showed no genomes. These findings indicate that PCR-ISH consistently detected a few copies of the EBV virions. The PCR-ISH was as sensitive as RNA-ISH. The RNA-ISH could not detect virus if RNA was not expressed, but the PCR-ISH could detect virus without such expression. The ability to detect a single copy of a specific gene in situ has many advantages and multiple applications in molecular biology, pathology, and cell biology.
Ohshima
K
K
Department of Pathology, School of Medicine, Fukuoka University, Japan.
Kikuchi
M
M
Kobari
S
S
Masuda
Y
Y
Yoneda
S
S
Takeshita
M
M
D001483:Base Sequence; D015139:Blotting, Southern; D004854:Herpesvirus 4, Human / Q000302:isolation & purification*; D006801:Humans; D017403:In Situ Hybridization; D008221:Lymphoid Tissue / Q000473:pathology* / Q000821:virology; D008223:Lymphoma / Q000473:pathology* / Q000821:virology; D008969:Molecular Sequence Data; D016612:Paraffin Embedding; D016133:Polymerase Chain Reaction; D014412:Tumor Virus Infections / Q000473:pathology*