Identification of lamivudine-resistant hepatitis B virus by oligonucleotide microarray

Abstract

Hepatitis B virus (HBV) is a partially double-stranded DNA virus that infects the human liver, causing cirrhosis and hepatocellular carcinoma. The nucleoside analogue lamivudine is an effective antiviral agent for HBV; however, drug-resistant HBV strains may emerge during prolonged lamivudine treatment. Lamivudine-resistant strains exhibit specific mutations, especially in the B and C domains of the HBV polymerase gene. The ability to detect such mutations is of increasing importance with increased availability of alternative antiviral agents such as penciclovir or famiciclovir, which show promise as a treatment for chronic HBV infections. We have developed a rapid and accurate HBV drug-resistant oligonucleotide chip for the detection of specific mutations in the HBV polymerase gene. Synthesized oligonucleotide probes of 25-32 bases in length with modifications at the 5'end were spotted in duplicate and covalently linked onto a glass slide. The oligonucleotide probes were hybridized with fluorescence-labeled complementary HBV DNA target molecules amplified from 40 samples that were confirmed to have single mutations by sequencing. This study shows that the results of the HBV drug-resistance oligonucleotide chip were identical to those of sequencing and reverse hybridization. The HBV drug-resistance oligonucleotide chip is very simple to use and provides an alternative method for the detection of HBV drug-resistance mutants.