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


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.
Bielawski
Krzysztof Piotr
KP
Molecular Diagnostics Division, Department of Biotechnology, Intercollegiate Faculty of Biotechnology, University of Gdańsk and Medical University of Gdańsk, Gdańsk, Poland. bielawski@biotech.ug.gda.pl
Al-Soud
Waleed Abu
WA
Stalke
Piotr
P
Charmuszko
Urszula
U
Wadstrom
Torkel
T
D020816:Amino Acid Motifs; D019380:Anti-HIV Agents / Q000494:pharmacology*; D001331:Automation; D001483:Base Sequence; D024882:Drug Resistance, Viral; D004586:Electrophoresis; D005240:Feasibility Studies; D015684:Gene Products, pol / Q000378:metabolism; D016679:Genome, Viral; D005838:Genotype; D006509:Hepatitis B / Q000175:diagnosis / Q000821:virology*; D006801:Humans; D019259:Lamivudine / Q000494:pharmacology*; D008969:Molecular Sequence Data; D009154:Mutation
D019380:Anti-HIV Agents; D015684:Gene Products, pol; C066027:P protein, Hepatitis B virus; D019259:Lamivudine