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 percent) with respect to mutation detection and genotyping. Complete agreement between the two methods was observed for all the samples (100 percent) at codon 180 and codon 204. Concordant results were observed for 99.4 percent of the 158 samples at codon 181 and 98.7 percent 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 percent 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.
Subject(s)
Humans , Adenine/analogs & derivatives , Antiviral Agents/pharmacology , Drug Resistance, Multiple, Viral/genetics , Hepatitis B virus/drug effects , Lamivudine/pharmacology , Mutation/genetics , Nucleosides/pharmacology , Phosphorous Acids , Pyrimidinones/pharmacology , Adenine/pharmacology , DNA, Viral/genetics , Genotype , Hepatitis B virus/genetics , Hepatitis B/virology , Ligase Chain Reaction , Microbial Sensitivity Tests , Multiplex Polymerase Chain ReactionABSTRACT
We describe a modified single nucleotide polymorphism (SNP) typing method based on the restriction fragment length polymorphism polymerase chain reaction (RFLP-PCR). This is a simple, economical method without the need for special equipment. For most SNP loci, a common restriction endonuclease (Hind III, EcoR I or BamH I) recognizing site (RER) can be introduced into one allelic form, but not the other by two rounds of mismatched PCR. The flanking regions can be changed by as many as five bases after PCR amplification with specially designed mismatching primers so the genotypes can be distinguished after digestion of the PCR products with corresponding endonucleases.