Hepatitis B virus quantification and detection of YMDD mutants in a single reaction by real-time PCR and annealing curve analysis

Yi Hsien Shih, Shiou Hwei Yeh, Pei Jer Chen, Wen Pin Chou, Hurng Yi Wang, Chun Jen Liu, Su Feng Lu, Ding Shinn Chen

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9 引文 斯高帕斯(Scopus)

摘要

Background: Long-term antiviral therapy, although effective for treatment of hepatitis B, might select the emergence of drug-resistant hepatitis B virus (HBV) mutants. Detection of HBV mutants and determination of viral titre are two crucial parameters for monitoring treatment response and occurrence of mutants. In this study, we take lamivudine resistance as an example to develop a method that can determine both parameters in a single-tube PCR reaction. Methods: The method contained two consecutive steps: in the first step, real-time PCR was used for quantification; in the second step, a novel annealing curve analysis was used for detecting YMDD mutants. For accurate quantification, PCR primers and hybridization probes were chosen from highly conserved regions to ensure the equivalent amplification of all HBV genotypes. Within the sensor probe, there were signature nucleotide polymorphisms that could effectively differentiate YMDD mutants from wild type by distinct melting temperatures (Tm) values. The clinical applicability of the assay was tested in serial samples from 90 patients receiving lamivudine treatment. Results: This assay could readily differentiate YMDD, YIDD and YVDD mutants by their distinct T m values. The quantification results showed great consistency in a linear range from 103 to 1011 copies/ml. Moreover, this assay could detect YMDD mutants accounting for ≤10% of the total viral population. Its clinical feasibility has been verified in primary specimens. Conclusions: The newly designed YMDD detection method is simple, sensitive, cost-effective, time-saving and provides a useful tool for follow-up of patients treated with lamivudine or other antiviral drugs.

原文英語
頁(從 - 到)469-480
頁數12
期刊Antiviral Therapy
13
發行號4
出版狀態已發佈 - 1月 1 2008
對外發佈

ASJC Scopus subject areas

  • 藥理
  • 藥學(醫學)
  • 傳染性疾病

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