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Journal of Clinical Microbiology, June 2005, p. 2810-2815, Vol. 43, No. 6
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.6.2810-2815.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Matrix-Assisted Laser Desorption Ionization-Time of Flight (Mass Spectrometry) for Hepatitis C Virus Genotyping

Elena N. Ilina,1* Maja V. Malakhova,1 Edward V. Generozov,1 Eugene N. Nikolaev,2 and Vadim M. Govorun1,3

Research Institute of Physical and Chemical Medicine, RF Health Ministry, Moscow, Russia,1 Institute for Energy Problems of Chemical Physics RAS, Moscow, Russia,2 Institute of Biomedical Chemistry RAMS, Proteomic Research Department, Moscow, Russia3

Received 24 June 2004/ Returned for modification 18 September 2004/ Accepted 12 February 2005

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.

* Corresponding author. Mailing address: Research Institute of Physical and Chemical Medicine, RF Health Ministry, Malaya Pyrogovskaya str, 1a, Moscow, 119992, Russia. Phone: (095) 245-4236. Fax: (095) 246-4501. E-mail: elena.ilina{at}lytech.ru.

Journal of Clinical Microbiology, June 2005, p. 2810-2815, Vol. 43, No. 6
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.6.2810-2815.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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