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.