Improved rolling circle amplification (RCA) of hepatitis B virus (HBV) relaxed-circular serum DNA (RC-DNA).
For functional analysis of HBV isolates, epidemiological studies and correct identification of recombinant genomes, the amplification of complete genomes is necessary. A method for completely in vitro amplification of full-length HBV genomes starting from serum RC-DNA is described. This uses in vitro completion/ligation of plus-strand HBV RC-DNA and amplification using Rolling-Circle Amplification, eventually followed by a genomic PCR. The method can amplify complete HBV genomes from sera with viral loads ranging from >1.0E+8 IU/ml down to 1.0E+3 IU/ml. The method can be applied to archived sera that have undergone long-term storage or to archived DNA serum extracts. The genomes can easily be cloned. HBV genotypes A-G can all be amplified with no apparent problems. A recombinant subgenotype A3/genotype E genome was identified and fully sequenced.
Centre de Recherche en Cancérologie de Lyon (CRCL); INSERM, U1052; CNRS, UMR 5286; UCBL1, S_1052. 151 cours Albert Thomas, 69003 Lyon, France.
Nora
N
Martel
Selma A
SA
Gomes
Isabelle
I
Chemin
Christian
C
Trépo
Alan
A
Kay
D004279:DNA, Viral / Q000097:blood* / Q000235:genetics; D016679:Genome, Viral; D006515:Hepatitis B virus / Q000235:genetics* / Q000302:isolation & purification*; D006801:Humans; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D044967:Serum / Q000821:virology*; D014773:Virology / Q000379:methods*
D004279:DNA, Viral
HBV; HBV genotypes; Recombinant genomes; Rolling-Circle Amplification (RCA); Serum relaxed-circular DNA