Development of a Method for Screening and Genotyping of HCV 1a, 1b, 2, 3, 4, and 6 Genotypes

Abstract

The World Health Organization and the World Health Assembly recommended eradicating hepatitis as a public threat by 2030. The accurate genotyping of hepatitis C virus (HCV) is crucial to achieving this goal because it is vital for the selection of anti-HCV therapy required for complete cure of HCV infection. We report the development of a method for accurate genotyping of HCV 1a, 1b, 2, 3, 4, and 6 genotypes. The merits of the developed method for HCV genotyping include (i) requirement of a single polymerase chain reaction (PCR) primer set, (ii) room-temperature detection in 30 min after the PCR, (iii) no need of highly trained professionals, (iv) highly accurate HCV genotyping results afforded by highly specific DNA-DNA hybridization, and (v) probe sequences that can be used on other platforms.

Figure 1

(a) HCV RNA structure including open reading frame and 5′ UTR, 3′ UTR regions, (b) alignment of sequences of HCV genotypes 1a, 1b, 2, 3, 4, and 6 in the 5′ UTR.

Figure 2

Selection of probes for HCV1a and HCV1b. (a) alignment of HCV1a and 1b sequences indicating the probe region, (b) hybridization of HCV 1a and 1b PCR products with HCV1a and HCV1b probes with and without an artificial mutation, (c) and (d) fluorescence intensities for respective probes upon hybridization with respective PCR products.

Figure 3

Selection of probes for HCV2 and HCV3 genotyping. (a, b) Fluorescence intensities of HCV2 probes upon hybridization with PCR products of HCV 1a, 1b, 2, 3, 4, and 6. (c, d) Fluorescence intensities of HCV3 probes upon hybridization with PCR products of HCV 1a, 1b, 2, 3, 4, and 6.

Figure 4

Selection of probes for HCV4 and HCV6 genotyping. (a, b) Fluorescence intensities of HCV4 probes upon hybridization with PCR products of HCV 1a, 1b, 2, 3, 4, and 6. (c, d) Fluorescence intensities of HCV6 probes upon hybridization with PCR products of HCV 1a, 1b, 2, 3, 4, and 6.