Rapid detection of hepatitis B virus mutations using real-time PCR and melting curve analysis

Current methods of detecting hepatitis B virus (HBV) mutations are time consuming, labor intensive, and not suitable for screening large numbers of samples. In the present study, we documented the advantages of a system that exploits differences in thermal stability between perfect match and mismatch hybrids, and thereby distinguishes between wild-type and mutants. Hybridization probes were designed complementary to specific wild-type HBV sequences in surface (S), precore, and basal core promoter (BCP) regions of the HBV genome (nt 587, 1896, and 1762/1764, respectively). Two probes were designed for each mutation: anchor probes were 3' labeled with fluorescein and sensor probes, 5' labeled with LC-Red 640, and 3' phosphorylated. Temperatures for each probe melted from amplification products were then determined in a melting program. Sera from 12 patients, each containing identified HBV mutants (6 S-escape, 1 precore, 1 BCP, and 4 mixed precore and BCP), and 5 control sera from patients with wild-type virus were analyzed. Genomic sequences of mutant and wild-type viruses were confirmed by direct sequencing. Real-time polymerase chain reaction (PCR) with fluorescent hybridization probes accurately identified each mutant and wild-type genome. Melting temperatures obtained from probe-product duplexes for the 3 mutants were distinguished from wild-type (>4.0 degrees C, minimal) within 45 minutes. The sensitivity of the system was 100 copies/mL and as few as 5% of mutant among wild-type virus were detected. In conclusion, real-time PCR with fluorescent hybridization probes is a specific, sensitive, quantitative, and rapid means of detecting clinically relevant HBV mutants.