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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

Bielawski, Krzysztof Plotr; Abu Al-Soud, Waleed LU ; Stalke, Plotr; Charmuszko, Urszula and Wadström, Torkel LU (2008) In Medical Science Monitor 14(5). p.281-285
Abstract
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... (More)
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. (Less)
Please use this url to cite or link to this publication:
author
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
genotyping, viral drug resistance, hepatitis B, YMDD motif mutation
in
Medical Science Monitor
volume
14
issue
5
pages
281 - 285
publisher
Medical Science Monitor
external identifiers
  • wos:000256199200012
  • scopus:43549097814
ISSN
1643-3750
language
English
LU publication?
yes
id
b47939d5-212c-4b7b-8241-2aaaafafa333 (old id 1201712)
alternative location
http://www.medscimonit.com/fulltxt.php?ICID=855755
date added to LUP
2008-09-15 11:48:33
date last changed
2017-01-01 04:23:31
@article{b47939d5-212c-4b7b-8241-2aaaafafa333,
  abstract     = {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.},
  author       = {Bielawski, Krzysztof Plotr and Abu Al-Soud, Waleed and Stalke, Plotr and Charmuszko, Urszula and Wadström, Torkel},
  issn         = {1643-3750},
  keyword      = {genotyping,viral drug resistance,hepatitis B,YMDD motif mutation},
  language     = {eng},
  number       = {5},
  pages        = {281--285},
  publisher    = {Medical Science Monitor},
  series       = {Medical Science Monitor},
  title        = {Determination of lamivudine-resistant variants of hepatitis B virus by denaturing gradient gel electrophoresis: A novel approach to monitoring drug resistance},
  volume       = {14},
  year         = {2008},
}