Detection of YMDD mutants using universal template real-time PCR

• Published in: World journal of gastroenterology : WJG 2006-02, Vol.12 (8), p.1308-1311
• Main Authors: Wang, Rong-Sheng, Zhang, Hua, Zhu, Yu-Fen, Han, Bei, Yang, Zhi-Jun
• Format: Article
• Language: English
• Subjects: Antiviral Agents - pharmacology; Antiviral Agents - therapeutic use; Aspartic Acid - analysis; DNA Mutational Analysis; DNA, Viral - analysis; DNA, Viral - genetics; Drug Resistance, Viral - genetics; Hepatitis B virus - drug effects; Hepatitis B virus - genetics; Hepatitis B, Chronic - drug therapy; Hepatitis B, Chronic - virology; Humans; Lamivudine - pharmacology; Lamivudine - therapeutic use; Methionine - analysis; Mutation; Polymerase Chain Reaction - methods; Rapid Communication; Sensitivity and Specificity; Sequence Analysis, DNA; Tyrosine - analysis; 乙型肝炎病毒; 病毒感染; 突变体; 聚合酶链反应
• ISSN: 1007-9327; 2219-2840
• DOI: 10.3748/wjg.v12.i8.1308

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 △Ct〈3.5 （△Ct = 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 widetype 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.