OBJECTIVETo establish a sensitive, specific, simple and practicable method for identifying the two sub-genotypes (Ba and Bj) of genotype B isolates of hepatitis B virus (HBV) (HBV/ B).

METHODSThe entire nucleotide sequences of HBV/B and HBV/C were obtained from GenBank, compared and analyzed with DNAStar software. The specific primers for HBV/B (HB) and Ba (BA), Bj (BJ) were designed respectively. HB as HBV/B specific primer (sense) and HBAS-4V (designed by Japanese scientists Sugauchi et al) as a universal outer primer (antisense) were used in the first-round PCR. In the second-round PCR, HB was also used as sense primer while BA and BJ as inner primers (antisense) and they were added into a single tube for PCR reaction. The two sub-genotypes of HBV/B were identified according to the length of the PCR products. A total of 71 HBV DNA-positive serum samples were selected randomly from our laboratory, including 56 HBV/B samples identified by type-specified PCR method and 15 HBV/C samples identified by direct sequencing in preS and S Region (preS/S). All the 71 samples were detected with this semi-nested PCR method. A plasmid containing full length genomic DNA of HBV/Bj, was presented by Professor Kenji Abe as positive control of Bj. Then, the first-round PCR products of 15 HBV/B were randomly selected and sequenced directly, and their sequences were compared phylogenetically with the above known Ba and Bj sequences using Blast and DNAStar softwares to confirm the results of semi-nested PCR.

RESULTS56 HBV/B samples were all identified as HBV/Ba by our semi-nested PCR method. 15 randomly selected PCR products were all sequenced as HBV/Ba. All of the 15 HBV/C samples were negative.

CONCLUSIONA simple, rapid, sensitive and specific method for identifying sub-genotypes Ba and Bj was established with might be used for large-scale clinical and epidemiological studies.