For one sample the LiPA method yielded an equivocal result of genotype 2 while sequences in both directions matched genotype 3a. The sequence based genotyping results were consistent with LiPA results except for three samples, where a different result at the main genotype level was obtained. The length of the obtained sequences was approximately 300?bp for the majority of samples. The four samples where no genotyping result was obtained were all gel-negative amplification reactions. In total, 197 genotyping results were obtained, including five gel-negative samples. The sensitivity of the method was found adequate for genotyping of patient samples, whose viral loads are generally between 10 4–10 7 IU/mL.Īltogether 201 amplification products were sequenced ( Table 2), including nine samples where no amplification product was observed in agarose gel. This may be due to long storage time and multiple freeze-thaw cycles of the samples. Amplification with the established method did not directly correlate with viral load. Samples which remained negative in gel had viral loads of 52–2550 IU/ml. Distinct but weaker products were obtained from samples whose known viral loads were 1020–9810 IU/ml. Strong amplification products were generally obtained from samples whose viral loads were over 10 000 IU/ml. Using the optimized PCR conditions presented above, a total of 238 serum samples were amplified ( Table 2).

Reaction conditions were adjusted for the following parameters: annealing temperature, primer concentration, magnesium concentration, amount of template and number of amplification cycles. Out of all primers, only 5'UTR1F and 5'UTR1R were able to amplify all samples in our testing set, and were thus selected for further optimization. For initial primer testing, a set of eight samples representing all main genotypes and most common subtypes (1a, 1b, 2a, 2b, 3a, 4, 5a and 6) was used.

4peaks chromatogram to fasta