Detection of YMDD mutation using mutant-specific primers in chronic hepatitis B patients before and after lamivudine treatment

AIM： To develop a PCR assay using mutant-specific primers to detect mutation of tyrosine-methionineaspartate-aspartate （YMDD） motif of HBV to tyrosinevaline-aspartate-aspartate （YVDD） or tyrosine-isoleucineaspartate-aspartate （YIDD）. METHODS： Cloned wild-type and mutant HBV sequences were used as te...

Full description

Saved in:
Bibliographic Details
Published in:World journal of gastroenterology : WJG 2006-09, Vol.12 (33), p.5301-5305
Main Authors: Lee, Cha-Ze, Lee, Hsuan-Shu, Huang, Guan-Tarn, Yang, Pei-Ming, Sheu, Jin-Chuan
Format: Article
Language:English
Subjects:
Amino Acid Motifs
Antiviral Agents - metabolism
Cloning, Molecular
DNA Primers - chemistry
Genes, Viral
Hepatitis B - drug therapy
Hepatitis B - virology
Hepatitis B virus - genetics
Humans
Lamivudine - pharmacology
Mutation
Reverse Transcriptase Inhibitors - pharmacology
Sensitivity and Specificity
Viral Hepatitis
Viral Proteins - genetics
基因突变
天门冬氨酸盐
慢性乙型肝炎
Citations: Items that this one cites
Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary:AIM： To develop a PCR assay using mutant-specific primers to detect mutation of tyrosine-methionineaspartate-aspartate （YMDD） motif of HBV to tyrosinevaline-aspartate-aspartate （YVDD） or tyrosine-isoleucineaspartate-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 wildtype HBV using this assay. YMDD mutants were detected in 8 of 12 HBeAg-positive patients and 8 of 16 HBeAgnegative 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.
ISSN:1007-9327
2219-2840
DOI:10.3748/wjg.v12.i33.5301