Long PCR and its application to hepatitis viruses: amplification of hepatitis A, hepatitis B, and hepatitis C virus genomes

Abstract

In this study we amplified virtually the entire genomes of hepatitis A virus (a member of the Picornaviridae family), hepatitis B virus (a member of the Hepadnaviridae family), and hepatitis C virus (a member of the Flaviviridae family) by using the recently described technique of long PCR. In order to do this, we first demonstrated, using the lambda phage, that long PCR can be made highly sensitive and the sensitivity can be further enhanced by nested long PCR. We also showed, using tobacco mosaic virus as a model, that a reverse transcriptase reaction can be linked to a long PCR, enabling the nearly full-length amplification of the genomes of RNA viruses. We then applied these techniques to serial dilutions of titrated stocks of well-characterized strains of hepatitis A, B, and C viruses. We amplified the nearly full-length sequence of each of these viruses from a small number of viral genomes, demonstrating the sensitivity of the process.