Abstract
Genotypic testing based on subtype-specific amplification and population Sanger sequencing for two nonstructural (NS) protein-coding regions, the NS3/4A protease and the NS5B polymerase, of the hepatitis C virus (HCV) genome is described here. The protocols include the molecular steps for RNA extraction, one-step RT-PCR followed by inner PCR and population Sanger sequencing, to obtain the sequence information of the target regions from the clinical isolates of HCV subtypes 1a and 1b, which can be used to detect any sequence change in the viral genome as for example caused by the development of drug resistance in these two common viral targets.
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Acknowledgement
The authors would like to thank Stéphanie Dumont for her invaluable contribution to the assay development.
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Koletzki, D., Pattery, T., Fevery, B., Vanhooren, L., Stuyver, L.J. (2013). Amplification and Sequencing of the Hepatitis C Virus NS3/4A Protease and the NS5B Polymerase Regions for Genotypic Resistance Detection of Clinical Isolates of Subtypes 1a and 1b. In: Gong, E. (eds) Antiviral Methods and Protocols. Methods in Molecular Biology, vol 1030. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-484-5_12
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DOI: https://doi.org/10.1007/978-1-62703-484-5_12
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Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-483-8
Online ISBN: 978-1-62703-484-5
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