A Real-Time PCR Assay for the Quantification of Hepatitis B Virus DNA and Concurrent Detection of YMDD Motif Mutations


Agca H., Sayiner A. A. , Sengonul A., Simsek I., Akarsu M.

MIKROBIYOLOJI BULTENI, vol.45, no.4, pp.664-676, 2011 (Peer-Reviewed Journal) identifier identifier identifier

  • Publication Type: Article / Article
  • Volume: 45 Issue: 4
  • Publication Date: 2011
  • Journal Name: MIKROBIYOLOJI BULTENI
  • Journal Indexes: Science Citation Index Expanded, Scopus, TR DİZİN (ULAKBİM)
  • Page Numbers: pp.664-676

Abstract

Monitoring therapy in chronic hepatitis B patients receiving lamivudine therapy, is done by two different assays; determination of viral load and genotypic resistance. These methods are labor intensive and time consuming. It was aimed to develop an assay to quantitate hepatitis B virus (HBV) DNA in serum and detect YMDD (thyrosine, methionine, aspartate, aspartate) motif mutations in the same run. The assay was based on real-time polymerase chain reaction (Rt-PCR) with YMDD-specific hybridization probes. Determination of YMDD motif was done by melting temperature analysis. External standard curve was used for quantifying viral DNA, which was generated by standard sera (VQC S2220) including HBV-DNA between concentrations of 1000 to 3 million copies/ml. The assay was compared with commercial quantitative kit (Artus HBV RG PCR; Qiagen, Germany), commercial line prob assay (INNO-LiPA HBV DR v1.0; Innogenetics, Belgium) and direct DNA sequencing method. Thirty-eight serum samples obtained from 20 chronic hepatitis B patients (7 female, 13 male; age range: 27-70 years) treated with only lamivudine and were negative for HIV and HCV antigen and antibodies were tested in the study. The analytical sensitivity of the assay was found as 200 copies/ml, with a dynamic range of 1 x 10(3) to 3 x 10(7) copies/ml. PCR efficiency of the in-house assay was found to be 1.98. Comparison of log(10) HBV-DNA concentrations determined by the in-house and commercial quantitative kits showed a significant correlation (r = 0.681). Melting temperature (T-m) analysis was used for the YMDD motif determination and found to be 59.86 degrees C for YMDD, 56.34 degrees C for YVDD and 55.10 degrees C for YIDD. The results of the in-house assay, DNA sequencing and LiPA were concordant in samples with homogeneous virus population, and in-house assay could also detect the major type of YMDD motif in mixed viral populations The Rt-PCR method which was developed in this study is a rapid, accurate and reproducible method for quantifying HBV-DNA and detecting the predominant YMDD motif in the same run in two hours duration. It was concluded that this method may be a convenient tool for monitoring HBV-infected patients receiving lamivudine treatment.