N 2 Evaluation of a Modified Sequence-based Method for HCV Genotyping

R.PARKES, G.MCNABB, P.COOK, M.KRAJDEN, S.K.BYRNE. British Columbia Centre for Disease Control. Vancouver, B.C.

Objective: Genotyping of HCV is useful for epidemiological investigations and for predicting interferon response or disease progression. Sequence-based HCV genotyping using the Amplicor HCV PCR  test product has been previously described. In that study the number of sequencing failures was high. To reduce the failure rate, we have adapted this method by simplifying the purification of PCR product and using two sequencing primers. We now report the results of an evaluation of this modified method.

Methods: Eighty-nine PCR products from the Cobas Amplicor test were genotyped in a blinded manner using both DNA sequencing and the INNO-LiPa II method. Prior to cycle-sequencing the PCR product was purified using Microcon spin columns. Both forward and reverse sequencing was performed using an ABI 310 DNA sequencer.

Results: Eighty-four (94%) of the HCV PCR products were successfully genotyped by the modified sequencing method. The remaining five could not be sequenced and had levels of PCR product below that detectable by gel electrophoresis. We found no major genotype differences between the results of both methods although seven had subtype differences between 1a and 1b.   

Conclusions: The sequence-based genotyping method was effective for genotyping HCV. However, in spite of using both forward and reverse sequencing, this still suffers a higher failure rate than the INNO-LiPa II method.