Typing of hepatitis B virus genomes by a simplified polymerase chain reaction

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Abstract

The amplification of hepatitis B virus (HBV) DNA sequences in sera for molecular epidemiology of HBV is an important application of the polymerase chain reaction (PCR) with regard to HBV. To simplify the PCR for this purpose, the optimal concentrations of SDS and detergents for carrying out the proteinase K digestion and the amplification of DNA by Taq polymerase were evaluated. It was found that by using 1% deoxycholic acid as detergent for the proteinase K step and diluting the digest 10 times before carrying out the PCR, the phenol extraction of DNA became superfluous. The sensitivity of this procedure equalled that of PCR after phenol extraction on comparable amounts of serum.

Four pairs of oligonucleotide primers were compared for amplification of HBV DNA sequences in 48 sera previously subtyped with respect to hepatitis B surface antigen (HBsAg), and in eight sera with different genotypes of HBV, representing the subtypes of HBsAg P1 to P8, defined at an international meeting [Couroucé-Pauty et al.: “HBs Antigen Subtypes.” Basel: Karger, 1976]. Two primer pairs, selected from conserved regions in the X and S genes of HBV, gave a positive PCR with sera harbouring all the eight different strains of HBV, resulting in DNA fragments consistent with the sizes deduced from genome sequence data. Two other primer pairs were selected in order to discriminate genotypes with regard to differences between d/y and w/r strains. With these primer sets total agreement was obtained with previous results from subtyping with regard to d/y and w/r with all 48 sera, but more complex results were obtained with P1 to P8, which partially, however, confirm a recent grouping of HBV genomes based on genetic relatedness [Okamoto et al.: Journal of General Virology 69:2575, 1988].

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