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- Title
A novel primer-extension assay for the detection of a G to A mutation in the distal precore region of hepatitis B virus DNA.
- Authors
Chung; Han; Kim; Yeh; Kim; Lee; Chon; Moon; Kang; Park; Han, Kwang Hyub
- Abstract
The roles of genetic heterogeneity of the hepatitis B virus (HBV) precore gene in the pathogenesis of HBV infection are unclear. Various methods have been used to detect nucleotide (nt) 1896 precore mutants. We established a new primer-extension assay to facilitate the detection of these mutants. This assay is based upon the fact that there is no adenine in the distal precore region of wild-type HBV. Polymerase chain reaction (PCR)-amplified template DNA was denatured and annealed to the [γ-32P]-labelled primer. During primer extension in the presence of DNA polymerase and dCTP, dGTP, dTTP and ddATP, the reaction terminates if there is a nucleotide A. When mixtures of different ratios of wild-type and nt 1896 precore mutants were analysed in the primer-extension assay, correlation between the percentage known amounts and the percentage measured amounts of nt 1896 precore mutants was excellent (r2=0.9669). When the primer-extension assay and direct sequencing were compared in hepatitis B e antigen (HBeAg)-positive and -negative chronic active hepatitis B patients, the primer-extension assay detected a greater number of nt 1896 precore mutants than direct sequencing and thus most HBV infections were found to be mixed infections. In conclusion, the primer-extension assay is a reliable and sensitive method for the detection of nt 1896 precore mutants.
- Subjects
VIRUS diseases; HEPATITIS B virus; PATHOLOGY
- Publication
Journal of Viral Hepatitis, 1999, Vol 6, Issue 4, p305
- ISSN
1352-0504
- Publication type
Article
- DOI
10.1046/j.1365-2893.1999.00169.x