Centre de Recherche en Cancérologie de Lyon (CRCL); INSERM, U1052; CNRS, UMR 5286; UCBL1, S_1052. 151 cours Albert Thomas, 69003 Lyon, France.
J Virol Methods. 2013 Nov;193(2):653-9. doi: 10.1016/j.jviromet.2013.07.045. Epub 2013 Aug 5.
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.
为了对 HBV 分离物进行功能分析、进行流行病学研究以及正确鉴定重组基因组，有必要扩增完整基因组。本文描述了一种从血清 RC-DNA 开始，完全在体外扩增全长 HBV 基因组的方法。该方法使用体外补全/连接正链 HBV RC-DNA，并通过 Rolling-Circle Amplification 进行扩增，最终进行基因组 PCR。该方法可以从病毒载量从 >1.0E+8 IU/ml 到 1.0E+3 IU/ml 的血清中扩增完整的 HBV 基因组。该方法适用于经过长期储存的存档血清或存档 DNA 血清提取物。基因组可以轻松克隆。可以毫不费力地扩增出所有基因型 A-G 的 HBV。鉴定并完全测序了一个重组亚基因型 A3/基因型 E 基因组。
