Detection of hepatitis B virus precore stop codon mutants by selective amplification method: frequent detection of precore mutants in hepatitis B e antigen positive healthy carriers.
The precore region of hepatitis B virus (HBV) is indispensable for secretion of e antigen protein. Therefore, the precore stop codon mutants may play an important role in the process of e antigen seroconversion. However, the presence of the mutants in hepatitis B e antigen positive carriers has not been fully studied because of difficulties in detecting the mutants in the presence of large amounts of wild-type viruses. To overcome this, a sensitive method has been developed to detect the presence of G to A stop codon mutants at codon 28 of precore region. Primers for polymerase chain reaction (PCR) were devised to introduce restriction enzyme site Sty I for wild-type viruses and Dde I for the mutants. The amplification products with these primers were digested with Sty I to exclude the products from wild-type viruses. The remaining amplicon from precore mutants were re-amplified, and the presence of precore mutant was confirmed with Dde I digestion. The presence of precore mutants was examined in 61 HBV carriers by the method combining PCR and restriction enzyme digestion. Approximately 0.1% of precore mutant DNA among 10(6) copies of wild-type virus DNA was detectable by this method. The presence of the precore mutants was detected in seven of 10 (70%) e antigen positive asymptomatic carriers, and in 29 of 36 (81%) e antigen positive patients with chronic liver diseases, and in all 15 (100%) anti-e antibody positive patients with chronic liver diseases. This study revealed that a small amount of the precore mutants was present in the majority of HBV carriers.
First Department of Medicine, Chiba University School of Medicine, Japan.
S
S
Nakahori
O
O
Yokosuka
T
T
Ehata
W L
WL
Chuang
F
F
Imazeki
Y
Y
Ito
M
M
Ohto
D000328:Adult; D001483:Base Sequence; D002353:Carrier State / Q000821:virology*; D018388:Codon, Terminator / Q000235:genetics*; D004279:DNA, Viral / Q000235:genetics*; D005260:Female; D006509:Hepatitis B / Q000821:virology*; D006513:Hepatitis B e Antigens / Q000032:analysis*; D006515:Hepatitis B virus / Q000235:genetics*; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity
D018388:Codon, Terminator; D004279:DNA, Viral; D006513:Hepatitis B e Antigens