Detection of YMDD mutation using mutant-specific primers in chronic hepatitis B patients before and after lamivudine treatment
Journal
World Journal of Gastroenterology
Journal Volume
12
Journal Issue
33
Pages
5301-5305
Date Issued
2006
Abstract
Aim: To develop a PCR assay using mutant-specific primers to detect mutation of tyrosine-methionine-aspartate-aspartate (YMDD) motif of HBV to tyrosine-valine-aspartate-aspartate (YVDD) or tyrosine-isoleucine-aspartate-aspartate (YIDD). Methods: Cloned wild-type and mutant HBV sequences were used as templates to test the sensitivity and specificity of the assay. A variety of primer construction, primer concentration, dNTP concentration, and annealing temperature of primers were systematically examined. Pair primers specific to rtL180M and rtM204V were selected for YVDD detection. Primer specific to rtM204I with an additional 3′-penultimate base mismatched to both the mutant and wild-type sequence was selected for YIDD detection. We applied this assay to study YMDD mutants in 28 chronic hepatitis B patients before and after lamivudine treatment. Results: We could detect as little as 0.001%-0.00001% of mutant viruses coexisting in 108-109 copies of wild-type HBV using this assay. YMDD mutants were detected in 8 of 12 HBeAg-positive patients and 8 of 16 HBeAg-negative patients before lamivudine treatment. After treatment, two more patients in HBeAg-positive patients and seven more patients in HBeAg-negative patients developed YMDD mutations. Conclusion: We developed a highly sensitive and specific assay for detecting YMDD mutants. This assay can be applied to monitor chronic hepatitis B patients before and during lamivudine treatment. ? 2006 The WJG Press. All rights reserved.
SDGs
Other Subjects
aspartic acid; hepatitis B(e) antigen; isoleucine; lamivudine; leucine; methionine; mutant protein; parathion; tyrosine; valine; 3' untranslated region; article; base mispairing; clinical article; concentration (parameters); controlled study; gene construct; genetic selection; hepatitis B; Hepatitis B virus; human; molecular cloning; mutational analysis; patient monitoring; polymerase chain reaction; protein motif; sensitivity and specificity; sequence analysis; temperature measurement; virus mutant; wild type
Publisher
Baishideng Publishing Group Co
Type
journal article