A refined long RT-PCR technique to amplify complete viral RNA genome sequences from clinical samples: Application to a novel hepatitis C virus variant of genotype 6

Document Type

Article

Publication Date

2005

Abstract

The goal of this study was to adapt a long RT-PCR technique to amplify large PCR fragments from the genome of hepatitis C virus (HCV) isolates using clinical samples. This was done by using a reverse transcriptase devoid of RNase H activity and a mixture of two antibody-bound thermostable polymerases to combine the high processivity of Taq and the high fidelity of Pwo with its 3′ → 5′ exonuclease activity. Other modifications included gentle handling during RNA extraction, the absence of tRNA and random primers, a two-step reverse transcription procedure to optimize cDNA synthesis, and increasing the annealing temperature for primers. With this approach, the HCV-1 genome (nucleotides 35-9282) was amplified consistently as two overlapping fragments of 5344 and 4675 bp from a pooled chimpanzee plasma sample containing approximately 106 genome copies of HCV RNA/ml. Using the conditions that we identified, 96% of the complete genomic sequence of a distinct HCV genotype 6 variant (km45) was determined from less than 300 μl of serum. This method should prove useful for molecular, epidemiological and clinical studies of hepatitis C where samples are limited but complete virus sequence is required, for example, identifying mutational hot spots of HCV under specific clinical conditions. © 2005 Elsevier B.V. All rights reserved.

Publication Title

Journal of virological methods

Volume

126

Issue

42006

First Page

139

Last Page

148

Comments

This article was published in Journal of virological methods, Volume 126, Issue 42006, Pages 139-148.

The published version is available at http://dx.doi.org/10.1016/j.jviromet.2005.01.031.

Copyright © 2005 Elsevier.

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