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

Abstract

Objectives: 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. Conclusion: 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.

Advances in ligase chain reaction and ligation-based amplifications for genotyping assays: Detection and applications

“…LCR technique has been used to detect mutations in the precore (preC), the presurface (PreS) regions in HBV genome with high detection rate [135,136]. The rtA181 V/T and rtN236T mutations that cause Adefovir dipivoxil (ADV) drug resistance and hence treatment failure were also assessed [137,138]. The rtM204 V and rtM204I mutations that cause resistance to lamivudine drug were detected by QDs-DNA nanosesnors [139].…”