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 paraffinembedded 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 β‐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 β‐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 β‐globin gene was observed in all. Furthermore, in all 25 frozen non‐A, non‐B livers, β‐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. Thus all patients with chronic hepatitis B, but no patients with clinically diagnosed non‐A, non‐B hepatitis, had hepatitis B virus DNA in liver at a level of or above one copy per hepatocyte. (H EPATOLOGY 1991;13:167–171).