Ilina Elena N, Malakhova Maja V, Generozov Edward V, Nikolaev Eugene N, Govorun Vadim M
Research Institute of Physical and Chemical Medicine, RF Health Ministry, Malaya Pyrogovskaya str, 1a, Moscow, 119992, Russia.
J Clin Microbiol. 2005 Jun;43(6):2810-5. doi: 10.1128/JCM.43.6.2810-2815.2005.
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.
丙型肝炎病毒（HCV）基因型的测定已成为实验室实践中的标准程序。基因型分类有助于疾病预后评估，并有助于确定适当的治疗时长。已描述了超过10种HCV类型和70种亚型。在俄罗斯，最常见的亚型是1a、1b、2a和3a，4型和5型相对少见。检测的“金标准”是基因测序。然而，已经开发出多种其他检测方法，以提供更快速、更廉价的检测形式。本研究的目的是通过微测序，然后进行基质辅助激光解吸电离飞行时间（MALDI-TOF）质谱分析来确定HCV基因型。扩增HCV基因组5'非翻译区的片段。设计了三种寡核苷酸引物，以检测两组基因型特异性单核苷酸多态性。引物延伸反应使用改良的热稳定DNA聚合酶，并在双脱氧核苷存在的情况下进行。用MALDI-TOF质谱仪分析反应产物的分子量。通过记录预期分子量的颗粒来确定HCV基因型。该方法用于对HCV阳性血清或血浆中的HCV进行基因分型。在所检测的样本中确定了1a、1b、2a、3a和4型HCV。数据通过直接测序得到证实。因此，我们提出了一种基于微测序和质谱分析的新的准确、高效的HCV基因分型方法。
