title: Demonstration of hepatitis C virus RNA with in situ hybridization employing a locked nucleic Acid probe in humanized liver of infected chimeric mice and in needle-biopsied human liver.
abstract: Background. In situ hybridization (ISH) with high sensitivity has been requested to demonstrate hepatitis C virus (HCV) RNA in formalin-fixed, paraffin-embedded (FFPE) sections of the liver. Methods. ISH employing a locked-nucleic-acid- (LNA-)modified oligonucleotide probe and biotin-free catalyzed signal amplification system (CSAII) was applied to HCV-RNA detection in the liver tissue. Nested reverse-transcription polymerase chain reaction (RT-PCR) was performed for HCV genotyping using total RNA extracted from FFPE sections. The target tissues included FFPE tissue sections of humanized livers in HCV-infected chimeric mice (HCV genotypes 1a, 1b, and 2a and noninfected) and of needle-biopsied livers from HCV-infected patients. Results. HCV-RNA was demonstrated with the ISH technique in HCV-infected liver tissues from both chimeric mice and 9 (82%) of 11 patients with HCV infection. The HCV signals were sensitive to RNase. Nested RT-PCR confirmed the genotype in 8 (73%) of 11 livers (type 1b: 6 lesions and type 2a: 2 lesions). HCV-RNA was not identified in chronic hepatitis B lesions, fatty liver, autoimmune hepatitis, and hepatocellular carcinoma. Conclusion. ISH using the LNA-modified oligonucleotide probe and CSAII was applicable to detecting HCV-RNA in routinely prepared FFPE liver specimens.
authors.affiliation: Department of Pathology, Fujita Health University School of Medicine, Toyoake, Aichi 470-1192, Japan.
authors.forename: Kazuya
authors.initials: K
authors.lastname: Shiogama
authors.forename: Ken-Ichi
authors.initials: K
authors.lastname: Inada
authors.forename: Michinori
authors.initials: M
authors.lastname: Kohara
authors.forename: Hidemi
authors.initials: H
authors.lastname: Teramoto
authors.forename: Yasuyoshi
authors.initials: Y
authors.lastname: Mizutani
authors.forename: Takanori
authors.initials: T
authors.lastname: Onouchi
authors.forename: Yutaka
authors.initials: Y
authors.lastname: Tsutsumi