Detection of the rtA181V/T and rtN236T mutations associated with resistance to adefovir dipivoxil using a ligase detection reaction assay

Abstract

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.