Hepatitis C Virus Genotyping Using an Oligonucleotide Microarray Based on the NS5B Sequence

Abstract

ABSTRACT The genotype of the hepatitis C virus (HCV) is essential for determining treatment duration in clinical practice and for epidemiological and clinical studies. Currently, few genotyping assays that determine the HCV subtype are available. This report describes a microarray-based molecular technique for identifying the HCV genotype and subtype. It uses low-density hydrogel-based biochips containing genotype- and subtype-specific oligonucleotides based on the sequences of the NS5B region of the HCV genome. The biochip contains 120 oligonucleotides that identify genotypes 1 to 6 and 36 (1a, 1b, 1c, 1d, 1e, 2a, 2b, 2c, 2d, 2i, 2j, 2k, 2l, 2m, 3a, 3b, 3k, 4a, 4c, 4d, 4f, 4h, 4i, 4k, 4n, 4o, 4p, 4r, 4t, 5a, 6a, 6b, 6d, 6g, 6h, and 6k) subtypes. The procedure included amplification of a 380-nucleotide (nt) fragment of NS5B and its hybridization on the biochip. Tests on 345 HCV-positive samples showed that the assay agreed with NS5B sequencing 100% for the genotype and 99.7% for the subtype. The hybridization on the microarray and the NS5B sequence were in 100% agreement for identifying the most common subtypes, 1a, 1b, 4a, 4d, and 3a. This approach is a promising tool for HCV genotyping, especially for implementing the new anti-HCV drugs that require accurate identification of clinically relevant subtypes.