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 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.
Chung
J P
JP
Department of Internal Medicine, Institute of Gastroenterology, Yonsei University College of Medicine, Seoul, Korea.
Han
K H
KH
Kim
K S
KS
Yeh
B I
BI
Kim
S T
ST
Lee
K S
KS
Chon
C Y
CY
Moon
Y M
YM
Kang
J K
JK
Park
I S
IS
D000328:Adult; D001483:Base Sequence; D017931:DNA Primers; D004279:DNA, Viral / Q000032:analysis; D005260:Female; D006513:Hepatitis B e Antigens / Q000032:analysis; D006515:Hepatitis B virus / Q000235:genetics*; D019694:Hepatitis B, Chronic / Q000821:virology*; D006801:Humans; D008297:Male; D008875:Middle Aged; D008969:Molecular Sequence Data; D017354:Point Mutation; D016133:Polymerase Chain Reaction / Q000379:methods; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA
D017931:DNA Primers; D004279:DNA, Viral; D006513:Hepatitis B e Antigens