Efficient amplification and cloning of near full-length hepatitis C virus genome from clinical samples

Abstract

Long RT-PCR (LRP) amplification of RNA templates is sometimes difficult compared to long PCR of DNA templates. Among RNA templates, hepatitis C virus (HCV) represents an excellent example to challenge the potential of LRP technology due to its extensive secondary structures and its difficulty to be readily cultured in vitro. The only source for viral genome amplification is clinical samples in which HCV is usually present at low titers. We have created a comprehensive optimization protocol that allows robust amplification of a 9.1 kb fragment of HCV, followed by efficient cloning into a novel vector. Detailed analyses indicate the lack of potential LRP-mediated recombination and the preservation of viral diversity. Thus, our LRP protocol could be applied for the amplification of other difficult RNA templates and may facilitate RNA virus research such as linked viral mutations and reverse genetics.

Fig. 1

Amplification of 9.1 kb fragment of HCV genome from serum samples JLR3037 (lanes 2 and 3) and RJ (lanes 4 and 5) by using optimized LRP protocol. The PCR product was electrophoresed on a 0.8% Seakem GTG agarose gel (FMC BioProducts). Lane 1, negative control; lane 6, 1 kb DNA ladder (Fisher).

Fig. 2

Amplification of 9.1 kb fragment of HCV genome from additional serum samples with various HCV RNA levels, including samples LIV19 and LIV23. The PCR product was electrophoresed on a 0.8% Seakem GTG agarose gel (FMC BioProducts). Lane 2, negative control; 1ane 1, 1 kb DNA ladder (Fisher); lane 16, Lambda DNA/HindIII markers (Promega).

Fig. 3

Comparison of HCV HVR1 (27 aa) quasispecies profiles derived from either 1.38 kb or 9.1 kb amplicons. Dots indicate the identity to the top line of amino acid sequence. While there is no obvious difference for sample LIV19 (A), LIV23 (B) displays much distinct HVR1 quasispecies profiles from two sizes of amplicons, 1.38 kb and 9.1 kb, respectively.

Fig. 4

A representative neighbor-joining (NJ) tree constructed based on HCV E1 domain of 20 clones derived from 9.1 kb LRP product, which was amplified using mixed serum from samples LIV19 and LIV23. As expected, all clones are clustered into two groups, LIV19 and LIV23. There is no contradictory clustering for each clone in trees constructed with other seven domains, indicating the lack of LRP-mediated recombination.