Quantitation of hepatitis B lamivudine resistant mutants by real-time amplification refractory mutation system PCR.
OBJECTIVE
Lamivudine is an antiviral drug that is used to treat hepatitis B virus (HBV) infection. Long-term therapy does not completely suppress viral replication, and resistant mutants emerge. Resistance is mediated by changes in the tyrosine-methionine-apartate-aspartate (YMDD) motif in the catalytic site of the HBV polymerase gene. We describe a method to detect and quantify mutant viral populations using amplification refractory mutation system (ARMS) PCR.


METHODS
We developed a real-time ARMS-PCR to detect point mutations in the polymerase gene. Using real-time PCR (LightCycler) with a ResonSense probe, PCRs were performed using clones of the HBV polymerase gene containing the different YMDD mutations. Dilution series of the templates were made and tested against each of the primer pairs. This method was applied to quantify mutant virus in patient serum samples.


RESULTS
As little as 0.01% mutant DNA in 10(5)-10(9) copies wild-type DNA were detected. The method is more sensitive than amplicon sequencing, which is the current method of mutant determination in the YMDD motif.


CONCLUSIONS
This study demonstrates a rapid, highly sensitive and reproducible method of quantifying mutant HBV virus in lamivudine treated patients. It can be used to monitor patients before and during lamivudine therapy.
Punia
Parvinder
P
Genomics Proteomics and Bioinformatics Unit, Health Protection Agency, 61 Colindale Avenue, London NW9 5HT, UK.
Cane
Patricia
P
Teo
Chong-Gee
CG
Saunders
Nicholas
N
D000595:Amino Acid Sequence; D001483:Base Sequence; D003062:Codon / Q000235:genetics; D017931:DNA Primers; D004279:DNA, Viral / Q000235:genetics* / Q000302:isolation & purification; D004259:DNA-Directed DNA Polymerase / Q000235:genetics; D024882:Drug Resistance, Viral; D006515:Hepatitis B virus / Q000187:drug effects / Q000235:genetics*; D019259:Lamivudine / Q000494:pharmacology*; D008969:Molecular Sequence Data; D009154:Mutation; D010446:Peptide Fragments / Q000737:chemistry; D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000379:methods*
D003062:Codon; D017931:DNA Primers; D004279:DNA, Viral; D010446:Peptide Fragments; D019259:Lamivudine; D004259:DNA-Directed DNA Polymerase