Evaluation of fluorescent hybridization biprobe PCR and melting curve assay

AIM: To evaluate a fluorescent hybridization biprobe PCR and melting curve assay for detection of (hepatitis B virus) YMDD mutation associated with lamivudine therapy. METHODS: HBV DNA and YMDD mutations in the 217 clinical serum specimens from patients with chronic HBV infection who were treated with lamivudine (100 mg/day) were detected by the fluorescence quantitative polymerase chain reaction (PCR) using TaqMan probe (FT-PCR) and the fluorescent hybridization biprobe PCR and melting curve assay (FH-PCR-MC), respectively. Seventy-eight positive sera were then genotyped by nested PCR with six pairs of HBV genotype-specific primes (A to F), and the cloned DNA fragments derived from conventional PCR of HBV YMDD of 30 positive sera were sequenced. (6/147). In HBV DNA 10 7 copies/L, all the positive and mutant rates of YMDD have no significant difference in different HBV DNA levels. Among 78 genotype samples (genotype C 89.8%, B 8.9% and D 1.3%), the positive and mutant rates of YMDD were 100%(78/78) and 58.97%(46/78) respectively. One genotype D was YIDD/YVDD. The mutant rates of YMDD in genotype B and C were 71.4% and 55.7% respectively, but have no marked difference (P >0.5). Using the results by DNA sequencing as reference standard, the relative specificity, sensitivity and over-all accordance of FH-PCR-MC were 96.3% (26/27), 100% (3/3) and 96.7% (29/30) respectively. The results of YMDD typing by FH- PCR-MC were confirmed by the sequencing of clones. CONCLUSION: The fluorescent hybridization biprobe PCR and melting curve assay kit in detection of HBV YMDD mutation has high sensitivity and specificity. It is a conve- nient and rapid test kit, and may be used in YMDD genotyping.