A novel primer-extension assay for the detection of a G to A mutation in the distal precore region of hepatitis B virus DNA

The roles of genetic heterogeneity of the hepatitis B virus (HBV) precore g ene 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. Th is assay is based upon the fact that there is no adenine in the distal prec ore region of wild-type HBV, Polymerase chain reaction (PCR)-amplified temp late DNA was denatured and annealed to the [gamma-P-32]-labelled primer. Du ring primer extension in the presence of DNA polymerase and dCTP, dGTP, dTT P and ddATP, the reaction terminates if there is a nucleotide A. When mixtu res of different ratios of wild-type and nt 1896 precore mutants were analy sed in the primer-extension assay, correlation between the percentage known amounts and the percentage measured amounts of nt 1896 precore mutants was excellent (r(2) = 0.9669), When the primer-extension assay and direct sequ encing were compared in hepatitis B e antigen (HBeAg)-positive and -negativ e 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.