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

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.