Matrix-assisted laser desorption ionization-time of flight (mass spectrometry) for hepatitis C virus genotyping

Abstract

Determination of the hepatitis C virus (HCV) genotype has become accepted as the standard procedure in laboratory practice. Genotype assignment helps in disease prognosis and assists in establishing the appropriate duration of treatment. More than 10 types and 70 subtypes of HCV have been described. In Russia the most common subtypes are 1a, 1b, 2a, and 3a, and the types 4 and 5 are relatively rare. The "gold standard" for testing is gene sequencing. However, a variety of other assays had been developed to provide more rapid and cheaper forms of testing. The aim of this study was to determine the HCV genotype by minisequencing followed by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. Fragments of 5' untranslated region of the HCV genome were amplified. Three oligonucleotide primers were designed to detect two sets of genotype-specific single nucleotide polymorphisms. The primer extension reaction was performed using modified thermostable DNA polymerase and in the presence of dideoxynucleosides. The molecular weights of the reaction products were analyzed with MALDI-TOF mass spectrometer. The HCV genotype was determined by registering the particles of the expected molecular weights. The method was used to genotype HCV from HCV-positive blood sera or plasma. The 1a, 1b, 2a, 3a, and 4 genotype HCVs were determined in the samples examined. The data were confirmed by direct sequencing. Thus, we propose a new accurate and efficient method for HCV genotyping based on minisequencing followed by mass spectrometry.

FIG. 1.

Alignment of the nucleotide sequences of the analyzed 5′ UTR fragments of HCV RNA belonging to various genotypes. The sites of annealing of the nucleotide primers participating in the minisequencing reaction are given in italics and underlined. The points of nucleotide polymorphisms are in bold capital letters.

FIG. 2.

Mass spectra of the minisequencing reaction products of the analyzed HCV genotypes. A. Nonextended oligonucleotide primers. B. The products of the primer extension in analysis of HCV genotype 1a. C. The products of the primer extension in analysis of HCV genotype 1b. D. The products of the primer extension in analysis of HCV genotype 2a. E. The products of the primer extension in analysis of HCV genotype 3a. F. The products of the primer extension in analysis of HCV genotype 4.