[Establishment of a nested PCR to identify hepatitis B virus genotypes A-D and subgenotypes B1, B2, C1 and C2]

To establish a hepatitis B virus (HBV) nested PCR (nPCR) for detection of genotypes A-D and subgenotypes B1, B2, C1 and C2.

The entire HBV nucleotide sequences of genotypes A-H retrieved from GenBank were compared and analyzed by DNAStar software. The PCR primers were designed by Primer Premier 5.0 software, and the nPCR for genotyping HBV/A-D as well as subgenotyping B1, B2, C1 and C2 were established. There were 3 steps in the process:step 1 for genotypes B, D and subgenotypes C1, C2 with the amplification of Mix A; step 2 for genotype A with the amplification of Mix B; step 3 for subgenotypes B1 and B2 with the amplification of Mix C in the second-round PCR, based on the first-round amplification procedure. A total of 68 serum samples from patients with chronic HBV infection were detected by nPCR. 15 of 68 sera were selected randomly and their PCR products were directly sequenced to confirm the accuracy of the method.

Among 68 serum samples of patients with chronic HBV infection detected by the nPCR, 23.53% (16/68) were infected with B2, 11.76% (8/68) with C1, 48.53% (33/68) with C2, 1.47% (1/68) with D, 11.76% (8/68) with B2C2 mix strains, 1.47% (1/68) with C2D mix strains and 1.47% (1/68) with B2/C1/D mix strains. The sequencing analysis of the 15 serum samples had the same results as detected by nPCR.

nPCR is a simple, rapid method and able to detect genotypes A-D and subgenotypes B1, B2, C1 and C2 subtypes of HBV with both high sensitivity and specificity.