Real-time PCR detection of multiple lamivudine-resistant mutations with displacing probes in a single tube.

BACKGROUND: Detection of lamivudine-resistant hepatitis B virus (HBV) is essential to clinical diagnosis and treatment. OBJECTIVES: To establish a single tube, real-time PCR assay for simultaneous detection of multiple lamivudine-resistant mutations in serum samples. STUDY DESIGN: By using four sequence-specific displacing probes labeled with different fluorophores, a single real-time PCR reaction can tell whether a sample contains any of the following HBV variants: wild-type, rtM204 mutant; mixtures of wild-type and rtM204 mutant; mixtures of rtM204 and rtL180 mutant; mixtures of wild-type, rtM204 mutant and rtL180 mutant. The assay was evaluated with 50 HBV mutation(s)-containing samples and 36 HBeAg-positive samples. RESULTS: The results of the real-time PCR assay were consistent with the DNA sequencing, but with much higher sensitivity for detecting a mixture of quasispecies. As few as 10(2)-10(3)copies/ml HBV of all four sequences in pure population and as little as 5% mutant DNA in the presence of wild-type DNA can be detected. CONCLUSIONS: Application of this high throughput assay into clinical use should enable earlier diagnosis and better treatment of lamivudine-resistant HBV.