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Open Access Methodology

Development of a sensitive RT-PCR method for amplifying and sequencing near full-length HCV genotype 1 RNA from patient samples

Eileen Z Zhang, Doug J Bartels, J Dan Frantz, Sheila Seepersaud, Judith A Lippke, Benjamin Shames, Yi Zhou, Chao Lin, Ann Kwong and Tara L Kieffer*

Author Affiliations

Vertex Pharmaceuticals Incorporated, 130 Waverly Street, Cambridge, MA, 02139-4242, USA

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Virology Journal 2013, 10:53  doi:10.1186/1743-422X-10-53

Published: 12 February 2013

Abstract

Background

Direct-acting antiviral (DAAs) agents for hepatitis C virus (HCV) span a variety of targets, including proteins encoded by the NS3/4A, NS4B, NS5A, and NS5B genes. Treatment with DAAs has been shown to select variants with sequence changes in the HCV genome encoding amino acids that may confer resistance to the treatment. In order to assess these effects in patients, a Reverse Transcription Polymerase Chain Reaction (RT-PCR) method was developed to sequence these regions of HCV from patient plasma.

Methods

A method was developed to amplify and sequence genotype 1 HCV RNA from patient plasma. Optimization of HCV RNA isolation, cDNA synthesis, and nested PCR steps were performed. The optimization of HCV RNA isolation, design of RT-PCR primers, optimization of RT-PCR amplification conditions and reagents, and the evaluation of the RT-PCR method performance is described.

Results

The optimized method is able to successfully, accurately, and reproducibly amplify near full-length genotype 1 HCV RNA containing a wide range of concentrations (103 to 108 IU/mL) with a success rate of 97%. The lower limit of detection was determined to be 1000 IU/mL HCV RNA.

Conclusions

This assay allows viral sequencing of all regions targeted by the most common DAAs currently in development, as well as the possibility to determine linkage between variants conferring resistance to multiple DAAs used in combination therapy.

Keywords:
Hepatitis C Virus (HCV); Reverse Transcription Polymerase Chain Reaction (RT-PCR); Genotype; Direct Acting Antiviral Agents (DAA); Lower Limit of Detection (LLOD); Quasispecies; Sequencing; Resistance; NS (Non-structural); NS3/4A; NS4B; NS5A; NS5B