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

作者：Chung JP; Han KH^*; Kim KS; Yeh BI; Kim ST; Lee KS; Chon CY; Moon YM; Kang JK; Park IS

来源：Journal of Viral Hepatitis, 1999, 6(4): 305-313.

DOI：10.1046/j.1365-2893.1999.00169.x

• chronic active hepatitis
• direct sequencing
• hepatitis B virus
• nt 1896 precore mutant
• primer-extension assay

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-P-32]-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 (r(2) = 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.

• 出版日期1999-7