Background: Adefovir dipivoxil (ADV) is effective for treatment of chronic hepatitis B virus (HBV) infection, but long-time ADV therapy leads to drug resistance because of HBV reverse transcriptase mutations. We developed a sensitive and specific method for detecting the rtA181V/T and rtN236T mutations associated with ADV resistance in chronic hepatitis B patients, based on a ligase detection reaction (LDR).
Methods: HBV templates were amplified by polymerase chain reaction (PCR), followed by LDR and electrophoresis on a sequencer. The assay was evaluated using 165 serum samples, and plasmid controls.
Results: In a mixture of wild-type and mutant plasmids, the assay could detect mutant plasmid at 1%. Complete concordance between the PCR-LDR assay and sequencing analysis was observed for 141 of 148 samples (95.3%) at codon 181, and 143 of 148 samples (96.6%) at codon 236. Discordant results were confirmed to be consistent with the PCR-LDR assay by subclone sequencing. Seventeen samples could not be detected by both of the methods due to low HBV DNA levels.
Conclusions: The PCR-LDR assay can sensitively and specifically detect the rtA181V/T and rtN236T mutations, and may be used for monitoring ADV resistance in patients infected with HBV.