High-resolution melting and real-time PCR for quantification and detection of drug-resistant HBV mutants in a single amplicon.
BACKGROUND
Antiviral therapy by nucleoside/nucleotide analogues (NAs) effectively reduces HBV replication in chronic hepatitis B (CHB) patients. Because long-term NA treatments will eventually select for drug-resistant mutants, early detection of mutants and frequent monitoring of viral loads is crucial for successful NA therapy. Because no efficient test for one-tube quantification and qualification of various HBV-resistant mutants exists, we propose to use high-resolution melting (HRM) analysis in combination with real-time PCR to achieve this unmet need.


METHODS
We developed a single amplicon for detecting HBV mutants resistant to lamivudine (LMV), adefovir (ADV) and entecavir (ETV), which are commonly used for CHB treatment. Our design consists of two steps: real-time PCR for viral quantification, and hybridization probe HRM analysis for detection of specific drug-resistant mutants.


RESULTS
Assay quantification was accurate (R=0.98) for viral loads from 10(3) to 10(9) copies/ml. HRM analysis produced distinct melting temperatures that clearly distinguished the mutants, rtM204V/I (LMV), rtA181V and rtN236T (ADV), and rtT184G and rtM250V (ETV), from their respective wild types. The assay detected mutants at only 10-25% of the HBV population. The clinical applicability of this assay was tested in a pilot study with serial samples from patients receiving LMV treatment.


CONCLUSIONS
Flexibility, speed and cost-efficiency are additional benefits unique to our assay. The clinical sample results further support the feasibility of applying our design to frequent and long-term monitoring of CHB patients receiving NA treatments in the clinical setting.
NTU Center for Genomic Medicine, National Taiwan University College of Medicine, Taipei, Taiwan.
Chia-Chieh
CC
Hsiao
Juliana
J
Chang
Jeng-You
JY
Wu
Wan-Hsin
WH
Liu
Shih-Yun
SY
Han
Pei-Jer
PJ
Chen
Shiou-Hwei
SH
Yeh
D000225:Adenine / Q000031:analogs & derivatives / Q000494:pharmacology / Q000627:therapeutic use; D000998:Antiviral Agents / Q000494:pharmacology / Q000627:therapeutic use; D018076:DNA, Complementary; D004279:DNA, Viral / Q000235:genetics; D024882:Drug Resistance, Viral / Q000235:genetics*; D006147:Guanine / Q000031:analogs & derivatives / Q000494:pharmacology / Q000627:therapeutic use; D006515:Hepatitis B virus / Q000187:drug effects* / Q000235:genetics*; D019694:Hepatitis B, Chronic / Q000175:diagnosis* / Q000188:drug therapy / Q000235:genetics*; D006801:Humans; D019259:Lamivudine / Q000494:pharmacology / Q000627:therapeutic use; D025202:Molecular Diagnostic Techniques; D009691:Nucleic Acid Denaturation; D063065:Organophosphonates / Q000494:pharmacology / Q000627:therapeutic use; D010865:Pilot Projects; D016133:Polymerase Chain Reaction / Q000379:methods; D019562:Viral Load
D000998:Antiviral Agents; D018076:DNA, Complementary; D004279:DNA, Viral; D063065:Organophosphonates; D019259:Lamivudine; C413685:entecavir; D006147:Guanine; C053001:adefovir; D000225:Adenine