[Detection of hepatitis B virus by using polymerase chain reaction and nonradioactive DNA probes. I. Identification of mutations in the precore region by PCR-direct sequencing and ASO probe method].
Seroconversion from hepatitis B e antigen (HBeAg) to anti-HBe antibody (anti-HBe) frequently occurs in hosts chronically infected with hepatitis B virus (HBV). Further, this phenomenon is related to a point mutation from guanine to adenine at nucleotide 83 in the precore region of HBV, which, converts codon 28 for tryptophan (TGG:W) to a translational, stop codon (TAG:X). Therefore, we decided to examine HBV in sera from patients for mutations in the precore region by a simple allele-specific oligonucleotide (ASO) probe method. Direct sequencing was first performed on DNA fragments amplified by the polymerase chain reaction in order to establish whether there were mutations in the precore region. Subsequently, specific DNA probes were applied to detection of mutations in the precore region. Subsequently, specific DNA probes were applied to detection of mutations in the precore gene. Five unknown mutations (I10N, C12W, C14S, V17F and A19D), three known mutations (I9V, W28X and G29D) and for novel nucleotide insertions were identified in anti-HBe positive sera. By using seven nonradioactive probes, we could determine the mutations at codons 9, 28 and 29 in anti-HBe positive sera. The W28X mutation was found in anti-HBe positive but not in any of HBeAg positive sera. Meanwhile, wild-type strains of HBV were detectable in sera from patients who were positive to HBeAg or anti-HBe. This ASO probe assay could determine in a few days the mutations in the precore region of HBV, especially including the defect to prohibit the synthesis and secretion of HBeAg.
D000595:Amino Acid Sequence; D001483:Base Sequence; D018388:Codon, Terminator; D006515:Hepatitis B virus / Q000235:genetics* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D009154:Mutation; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction / Q000379:methods*
D018388:Codon, Terminator; D015345:Oligonucleotide Probes