Detection of hepatitis B virus DNA in paraffin-embedded liver tissues in chronic hepatitis B or non-A, non-B, hepatitis using the polymerase chain reaction.
We developed a polymerase chain reaction assay for the direct detection of hepatitis B virus in paraffin-embedded liver tissue and applied this assay to determine whether hepatitis B virus DNA exists in livers with chronic hepatitis non-A, non-B. Fifty five liver biopsy samples were studied: 11 from patients with HBeAg-positive chronic hepatitis (paraffin-embedded) and 44 from patients with chronic hepatitis non-A, non-B (21 paraffin-embedded; 25 fresh frozen). Thirty three (75%) of the non-A, non-B cases were positive for hepatitis C virus antibodies. Approximately 1 to 10 ng of DNA was extracted from the paraffin-embedded tissue and amplified using oligonucleotide (23-mer) primers specific for the S gene (positions 261 to 692). The beta-globin gene was used as an internal control for sensitivity because this is a single copy gene and allows for relative quantification. In each of the chronic hepatitis B livers, the expected 432-base-pair amplification product for hepatitis B virus DNA and beta-globin gene product were both detected. On the other hand, in the 21 paraffin-embedded chronic hepatitis non-A, non-B livers, no hepatitis B virus DNA was detected, although beta-globin gene was observed in all. Furthermore, in all 25 frozen non-A, non-B livers, beta-globin gene was observed, but no hepatitis B virus band was seen. The limit of detection of hepatitis B virus DNA by this method was estimated to be one genomic copy of hepatitis B virus DNA per cell.(ABSTRACT TRUNCATED AT 250 WORDS)
Shindo
M
M
Department of Biotechnology, Kyoto Institute of Technology, Japan.
Okuno
T
T
Arai
K
K
Matsumoto
M
M
Takeda
M
M
Kashima
K
K
Shimada
M
M
Fujiwara
Y
Y
Sokawa
Y
Y
D000328:Adult; D002908:Chronic Disease; D004279:DNA, Viral / Q000032:analysis*; D005260:Female; D006509:Hepatitis B / Q000378:metabolism* / Q000382:microbiology; D006515:Hepatitis B virus / Q000235:genetics*; D006526:Hepatitis C / Q000378:metabolism* / Q000382:microbiology; D006801:Humans; D008099:Liver / Q000737:chemistry* / Q000382:microbiology; D008297:Male; D008875:Middle Aged; D016133:Polymerase Chain Reaction
D004279:DNA, Viral