Hepatitis B virus quantification and detection of YMDD mutants in a single reaction by real-time PCR and annealing curve analysis.
BACKGROUND
Long-term antiviral therapy, although effective for treatment of hepatitis B, might select the emergence of drug-resistant hepatitis B virus (HBV) mutants. Detection of HBV mutants and determination of viral titre are two crucial parameters for monitoring treatment response and occurrence of mutants. In this study, we take lamivudine resistance as an example to develop a method that can determine both parameters in a single-tube PCR reaction.


METHODS
The method contained two consecutive steps: in the first step, real-time PCR was used for quantification; in the second step, a novel annealing curve analysis was used for detecting YMDD mutants. For accurate quantification, PCR primers and hybridization probes were chosen from highly conserved regions to ensure the equivalent amplification of all HBV genotypes. Within the sensor probe, there were signature nucleotide polymorphisms that could effectively differentiate YMDD mutants from wild type by distinct melting temperatures (Tm) values. The clinical applicability of the assay was tested in serial samples from 90 patients receiving lamivudine treatment.


RESULTS
This assay could readily differentiate YMDD, YIDD and YVDD mutants by their distinct Tm values. The quantification results showed great consistency in a linear range from 10(3) to 10(11) copies/ml. Moreover, this assay could detect YMDD mutants accounting for < or = 10% of the total viral population. Its clinical feasibility has been verified in primary specimens.


CONCLUSIONS
The newly designed YMDD detection method is simple, sensitive, cost-effective, time-saving and provides a useful tool for follow-up of patients treated with lamivudine or other antiviral drugs.
Shih
Yi-Hsien
YH
NTU Center for Genomic Medicine, National Taiwan University College of Medicine, Taipei, Taiwan.
Yeh
Shiou-Hwei
SH
Chen
Pei-Jer
PJ
Chou
Wen-Pin
WP
Wang
Hurng-Yi
HY
Liu
Chun-Jen
CJ
Lu
Su-Feng
SF
Chen
Ding-Shinn
DS
D000328:Adult; D020816:Amino Acid Motifs / Q000235:genetics*; D000998:Antiviral Agents / Q000494:pharmacology / Q000627:therapeutic use; D001483:Base Sequence; D024882:Drug Resistance, Viral / Q000235:genetics*; D005260:Female; D005838:Genotype; D006515:Hepatitis B virus / Q000145:classification / Q000187:drug effects / Q000235:genetics / Q000302:isolation & purification*; D019694:Hepatitis B, Chronic / Q000188:drug therapy / Q000821:virology; D006801:Humans; D019259:Lamivudine / Q000494:pharmacology / Q000627:therapeutic use; D008297:Male; D008875:Middle Aged; D008969:Molecular Sequence Data; D009154:Mutation; D016133:Polymerase Chain Reaction / Q000191:economics / Q000379:methods*; D012680:Sensitivity and Specificity; D044366:Transition Temperature
D000998:Antiviral Agents; D019259:Lamivudine