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

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 [gamma-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.

Development of a Molecular-Beacon Assay To Detect the G 1896 A Precore Mutation in Hepatitis B Virus-Infected Individuals

“…These findings are consistent with other reports that denote the high specificity and sensitivity of molecular beacons used to detect SNPs (2,24,26,32). By contrast, direct sequencing is limited in its ability to detect small quantities of a strain within a mixed population, thus leading to false-negative results (8,30). The overall prevalence of the 1896 PC mutation in the pediatric patients in this study with HBeAg-negative status was 55.5%.…”