Comparison of hepatitis B virus subtyping of d/y determinants by radioimmunoprecipitation assay and the polymerase chain reaction.
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.
Department of Medical Microbiology, University of Edinburgh, Scotland.
W J
WJ
Nicholson
S H
SH
Black
P
P
Simmonds
C W
CW
Chung
D
D
Aw
J F
JF
Peutherer
D001483:Base Sequence; D016384:Consensus Sequence; D004279:DNA, Viral / Q000032:analysis* / Q000302:isolation & purification; D006514:Hepatitis B Surface Antigens / Q000145:classification* / Q000235:genetics; D006515:Hepatitis B virus / Q000145:classification* / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012150:Polymorphism, Restriction Fragment Length; D011863:Radioimmunoassay / Q000379:methods*; D012689:Sequence Homology, Nucleic Acid
D004279:DNA, Viral; D006514:Hepatitis B Surface Antigens