Determination of hepatitis C virus genotype by direct sequence analysis of products generated with the Amplicor HCV test.
Consistent with other members of the family Flaviviridae, hepatitis C virus (HCV) demonstrates a high degree of sequence variation throughout the coding regions of its genome. However, there is a high degree of sequence conservation found within the 5' untranslated region (UTR) of the genome, making this region a target of choice for most nucleic acid amplification-based detection assays. In this study, the Amplicor HCV test, a commercially available assay which detects the 5'UTR, was used for the detection of HCV RNA in 669 serum samples obtained from a cohort of liver transplantation patients. Amplification products obtained from the HCV-positive cases were subjected to direct sequencing and genotyping based upon seven phylogenetically informative regions within the 5'UTR. Of the 669 specimens, 416 (62.2%) tested positive for the presence of HCV RNA. Of these, 372 (89.4%) specimens were successfully classified into 11 HCV genotypes and subtypes after computer-assisted analysis of the sequence data. Forty-four (10.6%) of the HCV RNA-positive specimens were not classifiable, the majority corresponding to low-titer specimens as determined by the Chiron Quantiplex HCV RNA 2. 0 assay. Additional comparative studies targeting the NS-5 region of the viral genome generally confirmed the accuracy and sensitivity of the 5'UTR-based classifications, with the exception of the misclassification of a small number of type 1a cases as type 1b. We conclude that although the high sequence conservation within the 5'UTR results in the misclassification of a small number of HCV subtypes, the overall gains of efficiency, the shorter turnaround time, the inclusion of contamination control measures, and the low rate of test failure compared to that of methods based on the NS-5 gene together constitute significant advantages over other techniques.
Division of Experimental Pathology, Mayo Clinic, Rochester, Minnesota 55905, USA.
J J
JJ
Germer
P N
PN
Rys
J N
JN
Thorvilson
D H
DH
Persing
D020121:5' Untranslated Regions / Q000235:genetics; D014644:Genetic Variation; D016679:Genome, Viral; D016174:Hepacivirus / Q000235:genetics* / Q000302:isolation & purification; D006526:Hepatitis C / Q000821:virology*; D006801:Humans; D008828:Microbiological Techniques; D012367:RNA, Viral / Q000032:analysis / Q000235:genetics
D020121:5' Untranslated Regions; D012367:RNA, Viral