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Using a double polymerase chain reaction a method was devised for detecting and subtyping hepatitis B virus DNA in serum samples. Primers from the S-gene were selected from the sequence analyses of five HBV HBsAg subtypes, to amplify HBV DNA and subtype for y specific DNA. Thirty-eight samples were subtyped for d and y determinants by radioimmunoprecipitation assay (RIPA) and the polymerase chain reaction (PCR). Subtyping by PCR and RIPA was in agreement in 100% of subtype y samples and 83.3% of subtype d, giving an overall correlation of 92.1%. As a third comparison, 12 amplified samples were digested by the restriction enzyme Sau 3A, which differentiates between subtypes y and d. The digest results agreed with PCR in 83.3% of the samples. In addition, we compared our standard phenol/chloroform extraction against a rapid one step method. The phenol/chloroform stage was found to be essential for the removal of nucleases and polymerase inhibitors present in sera.

Original publication

DOI

10.1002/jmv.1890360105

Type

Journal article

Journal

J Med Virol

Publication Date

01/1992

Volume

36

Pages

21 - 27

Keywords

Base Sequence, Consensus Sequence, DNA, Viral, Hepatitis B Surface Antigens, Hepatitis B virus, Humans, Molecular Sequence Data, Polymerase Chain Reaction, Polymorphism, Restriction Fragment Length, Radioimmunoassay, Sequence Homology, Nucleic Acid