Hepatitis C genotyping by denaturing high-performance liquid chromatography

Abstract

Determination of the hepatitis C virus (HCV) genotype for infected patients increasingly has become accepted as the standard of care. Genotype assignment helps in assessing disease prognosis and assists in establishing the appropriate duration of treatment. The great genetic diversity of HCV, with 11 major genotypes and >70 subtypes, contributes to the technical difficulty of genotype testing. While the "gold standard" for testing is nucleic acid sequencing, a variety of hybridization assays, including the line probe assay, have been developed to provide more rapid and accessible forms of testing. The aim of this study was to determine whether denaturing high-performance liquid chromatography (dHPLC) could be used as a clinical method for distinguishing HCV genotypes 1, 2, 3, and 4. A portion of the 5' untranslated region of the HCV genome was amplified by heminested multiplex reverse transcription PCR. The two amplicons then were analyzed by dHPLC analysis and compared to the genotypes determined by sequence analysis. After 115 specimens were analyzed as standards, 200 masked specimens (specimens whose identity was not known before testing) were analyzed to determine the concordance of the assay. The assay had a concordance of 96% at the genotype level and a concordance of 87% at the subtype level. However, the dHPLC method was not as accurate as other reported methods of HCV genotyping. This is the first time that HCV genotyping has been performed by dHPLC.

FIG. 1.

Locations and sequences of primers used in the HCV heminested multiplex RT-PCR. (A) Sequence of HCV 5′ UTR (9) around amplicons of interest, showing the positions of primers HCV5UTR01F, HCV5UTR02R, and KY78. The primer locations are outlined in the boxes, and the numbers above the sequences are nucleotide positions with respect to the starting nucleotide of the translated region.

FIG. 2.

dHPLC trace of HCV heminested multiplex RT-PCR products under nondenaturing conditions at 50°C. The HCV specimen was type 1b. The elution of nucleic acid is based upon size in this analysis, so the first peak, at 2.9 min, is the 45-bp amplicon, and the peak at 9.3 min is the 153-bp amplicon.

FIG. 3.

Representative dHPLC traces for each genotype and a no-template control. (A) Analysis of the 153-bp amplicon carried out at 63.4°C with a 11.5 to 13.75% (vol/vol) acetonitrile gradient. (B) Analysis of the 45-bp amplicon carried out at 55°C with a 7.5 to 10%(vol/vol) acetonitrile gradient.

FIG. 4.

dHPLC retention times for the 153- and 45-bp amplicons of the 5′ UTR of HCV. The data are from the specimens used as standards.