Detection of hepatitis B virus DNA in hepatitis B surface antigen-negative serum by polymerase chain reaction: evaluation of different primer pairs and conditions

The presence of hepatitis B virus (HBV) DNA was investigated by polymerase chain reaction (PCR) in the serum of twenty Brazilian blood donors. All sera were negative for hepatitis B surface antigen (HBsAg), 17 of them presented antibodies to the hepatitis B core antigen (anti-HBc) as the unique serological marker of HBV infection, and 3 were positive for antibodies to HBsAg (anti-HBs) and anti-HBc. PCR assays were carried out using different pairs of oligonucleotides designed from conserved sequences of C, pre-S and S regions of the HBV genome. First, all oligonucleotide pairs were tested in PCR using plasmids carrying HBV genome from ayw or adw strains as templates. One-round PCR assays were able to detect 100-25,000 molecules of plasmid DNA, depending on the oligonucleotide pair, while semi-nested PCR assays detected 10-1000 molecules. The frequency of HBV DNA-positive results with HBsAg-sera varied from 0% to 50% depending upon the PCR assay. The results indicated that a number of both isolated anti-HBc and anti-HBs+, anti-HBc+ samples contained HBV DNA at a very low concentration, neighboring the limit of detection.