A Novel Accurate ACRS-PCR Method With a Digestion Internal Control for Identification of Wild Type and YMDD Mutants of Hepatitis B Virus Strains

J Virol Methods. 2006 Nov;137(2):298-303. doi: 10.1016/j.jviromet.2006.07.008. Epub 2006 Sep 8.

Abstract

As a consequence of the point mutation in the YMDD motif of the hepatitis B virus (HBV) polymerase gene, lamivudine-resistant mutants have been reported in chronic hepatitis B patients who underwent lamivudine therapy. The objective of the study was to develop a novel accurate artificially created restriction site (ACRS) method with a digestion internal control for identification of YMDD, YIDD and YVDD HBV strains. Three conserved, specific and diagnostic primers introducing NdeI, SspI and AleI cleavage sites were designed in order to identify YMDD, YIDD and YVDD strains, respectively; while, their reverse primers also modified with the above recognition sites in order to enzyme correctness monitoring and false outcome avoiding. Thirty-two chronic hepatitis B patients who had taken lamivudine for 1-3 years and checked by the Inno-LiPA HBV DR kit, were evaluated by the ACRS method and then compared to sequencing data. The results of the ACRS method revealed the YMDD mutant strain in 20 patients, YMDD plus YIDD pattern in 1 patient, YMDD plus YVDD in 4 patients, the YIDD in 4 patients and mixed infection with each three strains in 1 patient. The sequencing and Inno-LiPA results were in agreement with the ACRS results. The novel ACRS method is a reliable, rapid and a cost-effective technique for determination of HBV strains with the wild type and YMDD mutant patterns.

Publication types

  • Comparative Study
  • Evaluation Study

MeSH terms

  • Adult
  • Aged
  • Antiviral Agents / pharmacology
  • Antiviral Agents / therapeutic use
  • DNA Primers / genetics
  • DNA, Viral / genetics*
  • Deoxyribonucleases, Type II Site-Specific / metabolism
  • Drug Resistance, Viral / genetics*
  • Female
  • Hepatitis B virus / genetics*
  • Hepatitis B virus / isolation & purification
  • Hepatitis B, Chronic / drug therapy
  • Hepatitis B, Chronic / virology*
  • Humans
  • Lamivudine / pharmacology
  • Lamivudine / therapeutic use
  • Male
  • Middle Aged
  • Mutation*
  • Polymerase Chain Reaction / methods*
  • Polymerase Chain Reaction / standards
  • Reference Standards
  • Reproducibility of Results
  • Sequence Analysis, DNA

Substances

  • Antiviral Agents
  • DNA Primers
  • DNA, Viral
  • Lamivudine
  • endodeoxyribonuclease NdeI
  • endodeoxyribonuclease SspI
  • Deoxyribonucleases, Type II Site-Specific