Detection of YMDD mutants using universal template real-time PCR.

Abstract

AIM:

To establish a rapid and accurate method for the detection of lamivudine-resistant mutations in hepatitis B virus and monitor of lamivudine resistance during lamivudine treatment in patients with chronic hepatitis B virus infection.

METHODS:

We established a real-time PCR method using a universal template and TaqMan probe to detect YMDD mutants. Variants of YVDD and YIDD were tested by individual reactions (reaction V and reaction I) and total hepatitis B viruses were detected in another reaction for control (reaction C). Results were determined by deltaCt < 3.5 (deltaCt = Ct of reaction V or I - Ct of reaction C). Clones of the HBV polymerase gene containing different YMDD mutations were tested. Serum samples from 163 lamivudine-treated patients with chronic hepatitis B virus infection were detected using this method and the results were confirmed by DNA sequencing.

RESULTS:

As many as 1000 copies per milliliter of wide-type plasmid were detected and nonspecific priming was excluded. In the 163 samples from patients treated with lamivudine, lamivudine-resistant mutations were detected in 51 samples.

CONCLUSION:

This universal real-time PCR is a rapid and accurate method for quantification of YMDD mutants of HBV virus in lamivudine-treated patients and can be used to monitor lamivudine-resistant mutations before and during lamivudine therapy.

• Authors: Rong-Sheng Wang, Hua Zhang, Yu-Fen Zhu, Bei Han, Zhi-Jun Yang
• Journal: World journal of gastroenterology (World J Gastroenterol) Vol. 12 Issue 8 Pg. 1308-11 (Feb 28 2006) ISSN: 1007-9327 [Print] United States
• PMID: 16534892 (Publication Type: Comparative Study, Evaluation Study, Journal Article)

Chemical References

• Antiviral Agents
• DNA, Viral
• Lamivudine
• Aspartic Acid
• Tyrosine
• Methionine

Topics

• Antiviral Agents (pharmacology, therapeutic use)
• Aspartic Acid (analysis)
• DNA Mutational Analysis
• DNA, Viral (analysis, genetics)
• Drug Resistance, Viral (genetics)
• Hepatitis B virus (drug effects, genetics)
• Hepatitis B, Chronic (drug therapy, virology)
• Humans
• Lamivudine (pharmacology, therapeutic use)
• Methionine (analysis)
• Mutation
• Polymerase Chain Reaction (methods)
• Sensitivity and Specificity
• Sequence Analysis, DNA
• Tyrosine (analysis)