Quantification and genotyping of hepatitis B virus in a single reaction by real-time PCR and melting curve analysis

Shiou Hwei Yeh, Ching Yi Tsai, Jia Horng Kao, Chun Jen Liu, Ti Jung Kuo, Ming Wei Lin, Wen Ling Huang, Shu Fen Lu, Jane Jih, Ding Shinn Chen, Pei Jer Chen

Research output: Contribution to journalArticlepeer-review

196 Scopus citations

Abstract

Both viral titer and genotype of hepatitis B virus (HBV) play critical roles in determining clinical outcome and response to antiviral treatment in hepatitis B patients. In this study, a method was developed to determine both parameters in a single-tube reaction. The method contains two consecutive steps, the first step used real-time PCR for quantification and second step used melting curve analysis for genotyping. For accurate quantification, the PCR primers and hybridization probes were selected from highly conserved regions to ensure the equivalent amplification and hybridization of all genotypes of HBVs. Within the sensor probe there exists signature single nucleotide polymorphisms (SNPs), which could effectively differentiate different HBV genotypes by showing different melting temperatures. The quantification results showed great consistency with the commercial assays in linear range from 102 to 1011 copies/ml. By comparison with the traditional restriction fragment length polymorphism (RFLP) methods, 99% of samples were accurately genotyped by current assay, and with a higher detection rate. In addition, this method can detect mixed HBV infections. Currently, this methodology can be applied to areas prevalent with HBV genotypes B and C, providing an efficient alternative for clinical diagnosis and large-scaled longitudinal studies.

Original languageEnglish
Pages (from-to)659-666
Number of pages8
JournalJournal of Hepatology
Volume41
Issue number4
DOIs
StatePublished - Oct 2004

Keywords

  • Genotyping
  • Hepatitis B virus
  • Quantification
  • Real-time PCR

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