Detection of the rtA181V/T and rtN236T mutations associated with resistance to adefovir dipivoxil using a ligase detection reaction assay.
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.
Wang
Yong-Zhong
YZ
Institute for the Study of Liver Diseases, The Third Hospital of Changzhou, 50 Lanling Road, Changzhou 213001, China.
Xiao
Jun-Hua
JH
Ruan
Li-Hua
LH
Zhang
Hui-Yin
HY
Chen
Min
M
Pu
Xiang-Ke
XK
Shen
Hong-Yu
HY
Wu
Guo-Xiang
GX
D000225:Adenine / Q000031:analogs & derivatives* / Q000494:pharmacology / Q000627:therapeutic use; D000998:Antiviral Agents / Q000494:pharmacology* / Q000627:therapeutic use; D024882:Drug Resistance, Viral / Q000235:genetics*; D006509:Hepatitis B / Q000188:drug therapy; D006515:Hepatitis B virus / Q000187:drug effects / Q000201:enzymology / Q000235:genetics; D006801:Humans; D008025:Ligases / Q000378:metabolism*; D009154:Mutation; D063065:Organophosphonates / Q000494:pharmacology* / Q000627:therapeutic use; D010957:Plasmids / Q000235:genetics; D012194:RNA-Directed DNA Polymerase / Q000235:genetics*
D000998:Antiviral Agents; D063065:Organophosphonates; D012194:RNA-Directed DNA Polymerase; D008025:Ligases; D000225:Adenine; C106812:adefovir dipivoxil