Intracellular localization of polymerase chain reaction (PCR)-amplified hepatitis C cDNA.
The cellular distribution of hepatitis C virus was examined in formalin-fixed, paraffin-embedded tissues by in situ localization of the polymerase chain reaction (PCR)-amplified viral cDNA. Of nine liver biopsies studied, five were from patients seropositive for hepatitis C, one was from a seronegative patient who had detectable hepatitis C cDNA by standard reverse transcriptase (RT) PCR, and the remaining three were from patients without evidence of hepatitis C infection. Sequences homologous to viral RNA were rarely identified in the hepatitis C cases by standard RNA-cDNA in situ hybridization, but PCR-amplified viral cDNA was detectable in many hepatocytes in a panlobular distribution and in scattered Kupffer cells in each of the six hepatitis C cases and none of the controls. The pattern was equivalent whether intracellular localization was done by in situ hybridization after the RT and PCR steps or if digoxigenin-labeled nucleotide was incorporated into the amplified product. No signal was evident in the positive biopsies if the RT step was omitted, if "nonsense" primers were used, or if RT in situ PCR was preceded by RNase digestion. The RT in situ PCR technique allows for the rapid detection of any RNA virus, even if it is present in low copy number, and permits direct morphological correlation.
Nuovo
G J
GJ
Department of Pathology, State University of New York (SUNY), Stony Brook 11794-8691.
Lidonnici
K
K
MacConnell
P
P
Lane
B
B
D001483:Base Sequence; D004247:DNA / Q000032:analysis*; D004279:DNA, Viral / Q000032:analysis*; D016174:Hepacivirus / Q000235:genetics*; D006801:Humans; D017403:In Situ Hybridization; D007425:Intracellular Membranes / Q000382:microbiology*; D008099:Liver / Q000382:microbiology*; D015335:Molecular Probes; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods; D012194:RNA-Directed DNA Polymerase; D014018:Tissue Distribution
D004279:DNA, Viral; D015335:Molecular Probes; D004247:DNA; D012194:RNA-Directed DNA Polymerase