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Determination of lamivudine-resistant variants of hepatitis B virus by denaturing gradient gel electrophoresis: A novel approach to monitoring drug resistance

På svenska

Author

  • Krzysztof Plotr Bielawski
  • Waleed Abu Al-Soud
  • Plotr Stalke
  • Urszula Charmuszko
  • Torkel Wadström

Summary, in English

Background: A DGGE-based assay for hepatitis B virus (HBV) drug-resistance monitoring was designed and checked for feasibility. It detects mutations within the YMDD motif related to lamivudine resistance. Material/Methods: The YMDD motif of HBV polymerase was amplified by the set of primers designed in this study. DGGE analysis of the amplicons was performed on 9% polyacrylamide gels containing a 20-40% gradient of urea plus formamide and electrophoresis was performed. DNA sequencing was performed using a standard protocol. Results: Based on the DGGE pattern of previously sequenced HBV variants, a reference ladder consisting of bands was constructed within and near the YMDD motif of HBV with excellent resolution. The genotypes of all the fragments included in the ladder were confirmed by sequencing after DGGE analysis. The flexibility of DGGE was demonstrated by the ability to add more bands to the migration ladder when new variants were discovered during the analysis of patient specimens. Clinical samples from HBV-infected patients were also used to demonstrate the performance of this approach. Conclusions: This preliminary feasibility study of HBV drug-resistance monitoring by means of DGGE shows the potential advantage of this approach for low-cost screening for viral drug resistance in clinical settings. The presented example can be extended to detect other mutations related to drug resistance in the HBV genome as well as other viruses.

Publishing year

2008

Language

English

Pages

281-285

Publication/Series

Medical Science Monitor

Volume

14

Issue

5

Document type

Journal article

Publisher

Medical Science Monitor

Topic

  • Microbiology in the medical area

Keywords

  • genotyping
  • viral drug resistance
  • hepatitis B
  • YMDD motif mutation

Status

Published

ISBN/ISSN/Other

  • ISSN: 1643-3750