A general method for nested RT-PCR amplification and sequencing the complete HCV genotype 1 open reading frame

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A general method for nested RT-PCR amplification and sequencing the complete HCV genotype 1 open reading frame

Publisher
BioMed Central
Publication Date
Dec 01, 2005
Source
PMC
Keywords
Disciplines
  • Biology
  • Medicine
License
Unknown

Abstract

1743-422X-2-88.fm ral ss BioMed CentVirology Journal Open AcceMethodology A general method for nested RT-PCR amplification and sequencing the complete HCV genotype 1 open reading frame Ermei Yao1, John E Tavis*1,2 and the Virahep-C Study Group Address: 1Department of Molecular Microbiology and Immunology, Saint Louis University School of Medicine, Saint Louis, Missouri 63104, USA and 2Saint Louis University Liver Center, Saint Louis University School of Medicine, Saint Louis, Missouri 63104, USA Email: Ermei Yao - [email protected]; John E Tavis* - [email protected]; the Virahep-C Study Group - [email protected] * Corresponding author Abstract Background: Hepatitis C virus (HCV) is a pathogenic hepatic flavivirus with a single stranded RNA genome. It has a high genetic variability and is classified into six major genotypes. Genotype 1a and 1b cause the majority of infections in the USA. Viral genomic sequence information is needed to correlate viral variation with pathology or response to therapy. However, reverse transcription- polymerase chain reaction (RT-PCR) of the HCV genome must overcome low template concentration and high target sequence diversity. Amplification conditions must hence have both high sensitivity and specificity yet recognize a heterogeneous target population to permit general amplification with minimal bias. This places divergent demands of the amplification conditions that can be very difficult to reconcile. Results: RT and nested PCR conditions were optimized independently and systematically for amplifying the complete open reading frame (ORF) from HCV genotype 1a and 1b using several overlapping amplicons. For each amplicon, multiple pairs of nested PCR primers were optimized. Using these primers, the success rate (defined as the rate of production of sufficient DNA for sequencing with any one of the primer pairs for a given amplicon) for amplification of 72 genotype 1a and 1b patient plasma samples averaged over 95% for all amplicons. In addition, two

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