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.


OBJECTIVE
To establish a single tube, real-time PCR assay for simultaneous detection of multiple lamivudine-resistant mutations in serum samples.


METHODS
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.
Wen
Huixin
H
The Key Laboratory of Cell Biology and Tumor Cell Engineering of the Ministration of Education, and the Molecular Diagnostics Laboratory, Department of Biomedical Sciences, School of Life Sciences, Xiamen University, Fujian, China.
Li
Qingge
Q
D000998:Antiviral Agents / Q000494:pharmacology*; D004279:DNA, Viral / Q000235:genetics; D024882:Drug Resistance, Viral / Q000235:genetics*; D006509:Hepatitis B / Q000175:diagnosis* / Q000821:virology; D006515:Hepatitis B virus / Q000187:drug effects* / Q000235:genetics*; D006801:Humans; D019259:Lamivudine / Q000494:pharmacology*; D009154:Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D044967:Serum / Q000821:virology; D013223:Statistics as Topic
D000998:Antiviral Agents; D004279:DNA, Viral; D019259:Lamivudine