New hepatitis C virus genotyping assay as a routine method by novel restriction fragment mass polymorphism.

Abstract

Backgrounds/Aims: LiPA or genotype specific PCR (GS-PCR) assay for HCV genotyping has been clinically useful for predicting response to antiviral therapy. However, these methods have some limitations to identify mixed HCV infection and/or particular subtypes. The aim of this study was to introduce a new method, restriction fragment mass polymorphism (RFMP), which can effectively identify genotypes of HCV.

Material/Methods: We designed the RFMP to identify genotype specific mass. After 5’-NCR amplification products were obtained by RT-PCR, nested PCR was done with primers designed to introduce type IIS restriction endonuclease recognition sequence (Fokl) ahead of subtype-specific variable motif of 5’ NCR region. The enzymatic cleavage of products leads to excision of several oligonucleotide fragments containing subtype-specific variable motifs. The mass of these fragments determined by MALDI-TOF MS were variable according to type-specific oligonucleotide sequence. The proportion of genotype/subtype fraction was determined using different ratios of each type mixture with RFMP. Sixty-eight patients with chronic HCV infection were included. The genotype/subtypes of all 68 patients were determined by RFMP method and among them 33 patients were determined by both RFMP and existing methods such as LiPA and GS-PCR.

Results: Using RFMP method, the prevalence of 1b, 2a and mixed were 48.5~, 11.8~ and 16.5%. LiPA or GS-PCR assays could not identify type 2c, but the result of RFMP has shown that its prevalence was 22%, and was the main subtype of genotype 2 in Korea instead of 2a. Mixed infections determined by RFMP were 2a+2c, 2a+2e, 1b+2a, 1b+2a+2e, 1b+1f, 1b+1c, 1b+2c and 3e. The sensitivity of HCV genotypes determined by RFMP was 100 copies/ml. Using RFMP, we could determine the relative ratio of the mixed infection even though minor genotype/subtype was composed up to 2% of total population. When we compared the genotype results between LiPA and/or GS-PCR methods, 22 of 33 patients showed concordant results. Eleven of 33 patients, however, showed discordant results.

Conclusions: RFMP, a novel new genotyping assay, is a sensitive method and can be applied to large scale screening. It is more useful for diagnosing mixed infection and relative ratio of virus in mixed HCV infection compared to LiPA or GS-PCR.