Detection of HBV resistant mutations related to lamivudine, adefovir and entecavir by reverse hybridization technique

• Published in: Zhonghua gan zang bing za zhi Vol. 18; no. 6; p. 414
• Main Authors: Liu, Yan-Chen, Zhang, Wen-Lu, Hu, Yuan, Zhao, Li, Lai, Guo-Qi, Hu, Jie-Li, Yang, Feng, Huang, Ai-Long
• Format: Journal Article
• Language: Chinese
• Published: China 01.06.2010
• Subjects: Amino Acid Substitution; Antiviral Agents - pharmacology; DNA, Viral - genetics; Drug Resistance, Viral - drug effects; Drug Resistance, Viral - genetics; Hepatitis B virus - drug effects; Hepatitis B virus - genetics; Hepatitis B, Chronic - virology; Humans; Mutation; Nucleic Acid Hybridization - methods
• ISSN: 1007-3418
• DOI: 10.3760/cma.j.issn.1007-3418.2010.06.005

To establish a method for simultaneous detection of HBV resistant mutations associated with three kinds of nucleoside analogues. According to 981 HBV complete sequences in GenBank, two pairs of conserved primers labeled with digoxigenin were synthesized to amplify the region of HBV reverse transcriptase. To detect non-synonymous amino acid substitutions associated with lamivudine, adefovir and entecavir, 26 specific oligonucleotide probes covering ten different codon positions, I169T, V173L/G, L180M, A181T/V, T184G, S202I/G, M204V/I, Q215S, N236T and M250V/I/L were synthesized and immobilized on nylon membranes charged positively. The oligonucleotide probes immobilized on nylon membranes were then hybridized with PCR products labeled with digoxigenin to detect three drug-resistant mutations. In order to observe specificity and accuracy of probes, HBV wild-type, resistant reference strains and patients serums were assayed by reverse hybridization technique, respectively. The specific probes of 10 codon positio