LONG PCR AND ITS APPLICATION TO HEPATITIS VIRUSES - AMPLIFICATION OF HEPATITIS-A, HEPATITIS-B, AND HEPATITIS-C VIRUS GENOMES

In this study we amplified virtually the entire genomes of hepatitis A virus (a member of the Picornaviridae family), hepatitis B virus (a m ember of the Hepadnaviridae family), and hepatitis C virus (a member o f the Flaviviridae family) by using the recently described technique o f long PCR In order to do this, we first demonstrated, using the lambd a phage, that long PCR can be made highly sensitive and that the sensi tivity can de further enhanced by nested long PCR, We also showed, usi ng tobacco mosaic virus as a model, that a reverse transcriptase react ion can be linked to a long PCR, enabling the dearly full-length ampli fication of the genomes of RNA viruses, We then applied these techniqu es to serial dilutions of titrated stocks of well-characterized strain s of hepatitis A, B, and C viruses, We amplified the nearly full-lengt h sequence of each of these viruses from a small number of viral genom es, demonstrating the sensitivity of the process.