Detection and direct sequencing of hepatitis B virus genome by DNA amplification method.
Hepatitis B virus (HBV) DNA was detected with amplification by the polymerase chain reaction method. Cloned HBV DNA equivalent to one virus genome (3 x 10(-6) pg) was detectable by ethidium bromide staining after 50 cycles of polymerase chain reaction. By applying this method, presence of HBV DNA was studied in 23 hepatitis B surface antigen (HBsAg)-positive and 11 HBsAg-negative sera from patients with chronic liver disease. Hepatitis B virus DNA was positive in 8 of 8 hepatitis B e antigen (HBeAg)-positive, in 2 of 2 HBeAg- and anti-HBe-negative, and in 12 of 13 anti-HBe-positive sera. Hepatitis B virus DNA was undetectable in all HBsAg-negative sera even with amplification. To confirm specificity, the amplified product was directly sequenced. Sequences around 122nd and 160th codon of HBs gene, which determines subtypes d/y and w/r, respectively, were analyzed. The results were compatible with recent reports regarding the relation between HBV subtypes and HBV DNA sequence at those codons. Hepatitis B virus DNA could be detected at the level of one virion by gene amplification method, and its sequence could be determined by direct sequencing in a few days.
Yokosuka
O
O
First Department of Medicine, Chiba University School of Medicine, Japan.
Omata
M
M
Hosoda
K
K
Tada
M
M
Ehata
T
T
Ohto
M
M
D001483:Base Sequence; D002908:Chronic Disease; D004279:DNA, Viral / Q000032:analysis* / Q000097:blood; D005814:Genes, Viral; D006515:Hepatitis B virus / Q000235:genetics*; D006801:Humans; D008107:Liver Diseases / Q000382:microbiology; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction
D004279:DNA, Viral