Figure 1
HBV genome and real-time PCR strategy. (A) HBV wild-type full genome with SP1 5’ splice site, 1.2 kb intron and 3’ splice site. (B) HBV genomes before (3.2 kb) and after (2.2 kb) the SP1 splicing. (C) Plasmid constructs carrying an insert (XbaI—EcoRI) of either HBV 5’ splice site (unspliced) or SP1 splice junction and the 5’ end of the X gene. The locations of real-time PCR primers and probes to detect the 5’ splice site, SP1 splice junction and X gene are shown. Using B-M54923 as an HBV reference sequence, the core gene forward primer (FPc) is located at nt 2365—2384, the core gene reverse FAM probe (PRc) is at nt 2428—2402, the core gene reverse primer (RPwt) designed to cross the 5’ splice site to specifically detect the unspliced wild-type HBV full genome is at nt 2465—2444, the shifted reverse primer (RPsp) designed to cross the SP1 splice junction region to specifically detect the spliced SP1 HBV genome is at nt 507—489 ^ 2447—2444, the X gene forward primer (FPx) is at nt 1528—1547, the X gene forward CY5 probe (PRx) is at nt 1561—1581, and the X gene reverse primer (RPx) is at nt 1600—1582.
