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Open Access Methodology

Quantitation of substitutions at amino acid 70 in hepatitis C virus genotype 1b

Zhongjie Hu*, Ying Liu, Lixia Qiu, Zuopeng Fan, Wei Nie, Shan Liang and Ronghua Jin

Author Affiliations

Department of Hepatitis C & Toxic liver diseases, Beijing Youan Hospital, Capital Medical University, No. 8 Xitoutiao, Youanmenwai, Fengtai District Beijing 100069, the People’s Republic of China

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Virology Journal 2014, 11:148  doi:10.1186/1743-422X-11-148

Published: 15 August 2014

Abstract

Background

Substitutions of amino acid (aa) 70 in the core region of hepatitis C virus genotype 1b (HCV 1b) are a predictor of the non-virological response to pegylated interferon plus ribavirin (PEG-IFN/RBV) therapy. The aim of our study was to develop quantitative real-time reverse transcription polymerase chain reaction (qPCR) assays to quantify wild-type (70 W) and mutant (70 M) strains of HCV 1b.

Methods

We used the TaqMan system to quantify strains 70 W and 70 M. Codon 70 in the HCV 1b core region can be either CGN or CAN, therefore degenerate TaqMan minor groove binder (MGB) probes with inosine were used. We determined detection limits, sensitivity and specificity of the methods developed. Direct sequencing and cloning of the HCV core region were used to confirm the reliability of our new system. Serum samples from 138 Chinese patients infected with HCV 1b were examined with the system we developed and compared with results obtained from the Roche TaqMan RT-PCR HCV RNA quantitation system.

Results

Degenerate MGB probes were able to clearly distinguish 70 W from 70 M. The detection limit was 103 copies/mL. Cross-reactivity tests confirmed the specificity of our method. Our system can effectively quantify 70 W and 70 M for 99.6% of patients with HCV 1b. Further tests involving cloning and sequencing confirmed the reliability of our system.

Conclusions

We developed an assay system using degenerate TaqMan MGB probes with inosine to quantify wild-type and mutant viral RNAs of the HCV 1b core region at aa 70. Our developed assay system had high levels of sensitivity and accuracy, and could prove useful in investigating dynamic changes during PEG-IFN/RBV therapy to assess virological responses.

Keywords:
Hepatitis C virus; Genotype 1b; Amino acid 70 substitutions; Degenerate probes; Inosine; Quantitative PCR