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Journal of Clinical Microbiology, October 2000, p. 3581-3584, Vol. 38, No. 10
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Genotyping of Hepatitis C Virus Isolates using CLIP Sequencing

R. S. Ross,1,* S. O. Viazov,1 C. D. Holtzer,2 A. Beyou,2 A. Monnet,2 C. Mazure,2 and M. Roggendorf1

Institute of Virology, National Reference Centre for Hepatitis C, University of Essen, D-45122 Essen, Germany,1 and Visible Genetics Europe, Genopole, F-91035 Evry Cedex, France2

Received 6 April 2000/Returned for modification 22 June 2000/Accepted 27 July 2000

Determination of hepatitis C virus (HCV) genotypes and subtypes has become increasingly important for the clinical management and prognosis of HCV infections. The aim of the present study was to assess the specificity and reliability of a newly developed, commercially available HCV genotyping kit (TRUGENE HCV 5'NC genotyping kit). This technique utilizes PCR fragments previously generated by the diagnostic Roche AMPLICOR HCV test, which are subsequently subjected to simultaneous PCR amplification and direct sequencing (CLIP sequencing) of the 5' noncoding region (5'NCR). HCV isolates from 100 randomly chosen patients were genotyped by both the TRUGENE HCV 5'NC genotyping kit and DNA enzyme immunoassay (DEIA). Typing results obtained by both methods were in complete concordance in 91% of the cases. HCV RNA from the samples with discordant genotype assignment in both assays was additionally amplified with primers from the HCV core and NS5B regions. Phylogenetic analysis of the obtained sequences supported the results obtained from DEIA in six cases and CLIP sequencing in two cases. In the former six cases, the TRUGENE HCV 5'NC genotyping kit could not correctly differentiate between subtypes of genotypes 1 and 2 due to the high conservation of the 5'NCR. However, since there was not any misclassification between HCV genotypes 1 and non-1 types, the results obtained with this system are, in general, reliable and can be used in clinical practice. The TRUGENE HCV 5'NC genotyping kit in our hands proved to be a fast and convenient technique that might be an attractive option for HCV genotyping in laboratories already using the Roche AMPLICOR HCV test for diagnostic reverse transcription-PCR.

* Corresponding author. Mailing address: Institute of Virology, National Reference Centre for Hepatitis C, University of Essen, Hufelandstr. 55, D-45122 Essen, Germany. Phone: 49 201 7233561. Fax: 49 201 7235929. E-mail: stefan.ross{at}uni-essen.de.

Journal of Clinical Microbiology, October 2000, p. 3581-3584, Vol. 38, No. 10
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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