Quantitation of hepatitis B lamivudine resistant mutants by real-time amplification refractory mutation system PCR

Parvinder Punia, Patricia Cane, Chong Gee Teo, Nicholas Saunders*

*Corresponding author for this work

    Research output: Contribution to journalArticlepeer-review

    43 Citations (Scopus)

    Abstract

    Background/Aims 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 105-109 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.

    Original languageEnglish
    Pages (from-to)986-992
    Number of pages7
    JournalJournal of Hepatology
    Volume40
    Issue number6
    DOIs
    Publication statusPublished - Jun 2004

    Keywords

    • Amplification refractory mutation system PCR
    • Detection of mutants
    • Hepatitis B virus
    • Lamivudine
    • Real-time PCR
    • YMDD

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