Simultaneous detection of hepatitis B virus genotypes and mutations associated with resistance to lamivudine, adefovir, and telbivudine by the polymerase chain reaction-ligase detection reaction.
OBJECTIVE
Detection of mutations associated to nucleos(t)ide analogs and hepatitis B virus (HBV) genotyping are essential for monitoring treatment of HBV infection. We developed a multiplex polymerase chain reaction-ligase detection reaction (PCR-LDR) assay for the rapid detection of HBV genotypes and mutations associated with lamivudine, adefovir, and telbivudine resistance in HBV-infected patients.


METHODS
HBV templates were amplified by PCR, followed by LDR and electrophoresis on a sequencer. The assay was evaluated using plasmids that contained wild-type or mutant HBV sequences and 216 clinical samples.


RESULTS
The PCR-LDR assay and sequencing gave comparable results for 158 of the 216 samples (73.1%) with respect to mutation detection and genotyping. Complete agreement between the two methods was observed for all the samples (100%) at codon 180 and codon 204. Concordant results were observed for 99.4% of the 158 samples at codon 181 and 98.7% at codon 236. The genotyping results were completely concordant between the PCR-LDR assay and sequencing. The PCR-LDR assay could detect a proportion of 1% mutant plasmid in a background of wild-type plasmid.


CONCLUSIONS
The PCR-LDR assay is sensitive and specific for detection of HBV genotypes and drug resistance mutations, and could be helpful for decision making in the treatment of HBV infection.
Institute for the Study of Liver Diseases, The Third People's Hospital of Changzhou, 300 North Lanling Road, Changzhou, Jiangsu Province, China. wyz213003@163.com
Yong-Zhong
YZ
Wang
Jun-Hua
JH
Xiao
Long-Gen
LG
Liu
Chun-Yan
CY
Ye
Hong-Yu
HY
Shen
Tian-Min
TM
Xu
Ke-Zhuan
KZ
Zhu
D000225:Adenine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000998:Antiviral Agents / Q000494:pharmacology*; D004279:DNA, Viral / Q000235:genetics; D024921:Drug Resistance, Multiple, Viral / Q000235:genetics*; D005838:Genotype; D006509:Hepatitis B / Q000821:virology; D006515:Hepatitis B virus / Q000187:drug effects* / Q000235:genetics; D006801:Humans; D019259:Lamivudine / Q000494:pharmacology*; D022521:Ligase Chain Reaction; D008826:Microbial Sensitivity Tests; D060885:Multiplex Polymerase Chain Reaction; D009154:Mutation / Q000235:genetics*; D009705:Nucleosides / Q000494:pharmacology*; D063065:Organophosphonates / Q000494:pharmacology*; D011744:Pyrimidinones / Q000494:pharmacology*; D000077712:Telbivudine; D013936:Thymidine / Q000031:analogs & derivatives
D000998:Antiviral Agents; D004279:DNA, Viral; D009705:Nucleosides; D063065:Organophosphonates; D011744:Pyrimidinones; D000077712:Telbivudine; D019259:Lamivudine; C053001:adefovir; D000225:Adenine; D013936:Thymidine