Liew Michael, Erali Maria, Page Sam, Hillyard David, Wittwer Carl
Institute for Clinical and Experimental Pathology, ARUP Laboratories, Salt Lake City, Utah 84108-122, USA.
J Clin Microbiol. 2004 Jan;42(1):158-63. doi: 10.1128/JCM.42.1.158-163.2004.
Determination of the hepatitis C virus (HCV) genotype for infected patients increasingly has become accepted as the standard of care. Genotype assignment helps in assessing disease prognosis and assists in establishing the appropriate duration of treatment. The great genetic diversity of HCV, with 11 major genotypes and >70 subtypes, contributes to the technical difficulty of genotype testing. While the "gold standard" for testing is nucleic acid sequencing, a variety of hybridization assays, including the line probe assay, have been developed to provide more rapid and accessible forms of testing. The aim of this study was to determine whether denaturing high-performance liquid chromatography (dHPLC) could be used as a clinical method for distinguishing HCV genotypes 1, 2, 3, and 4. A portion of the 5' untranslated region of the HCV genome was amplified by heminested multiplex reverse transcription PCR. The two amplicons then were analyzed by dHPLC analysis and compared to the genotypes determined by sequence analysis. After 115 specimens were analyzed as standards, 200 masked specimens (specimens whose identity was not known before testing) were analyzed to determine the concordance of the assay. The assay had a concordance of 96% at the genotype level and a concordance of 87% at the subtype level. However, the dHPLC method was not as accurate as other reported methods of HCV genotyping. This is the first time that HCV genotyping has been performed by dHPLC.
确定感染患者的丙型肝炎病毒（HCV）基因型日益被视为标准治疗手段。基因型分类有助于评估疾病预后，并协助确定适当的治疗时长。HCV具有高度的基因多样性，有11种主要基因型和70多种亚型，这增加了基因型检测的技术难度。虽然检测的“金标准”是核酸测序，但已开发出多种杂交检测方法，包括线性探针检测法，以提供更快速、更易操作的检测形式。本研究的目的是确定变性高效液相色谱法（dHPLC）是否可作为区分HCV 1、2、3和4型基因型的临床方法。通过半巢式多重逆转录PCR扩增HCV基因组5'非翻译区的一部分。然后通过dHPLC分析对两个扩增子进行分析，并与序列分析确定的基因型进行比较。在将115个标本作为标准进行分析后，对200个盲法标本（检测前身份未知的标本）进行分析以确定检测的一致性。该检测在基因型水平的一致性为96%，在亚型水平的一致性为87%。然而，dHPLC方法不如其他已报道的HCV基因分型方法准确。这是首次使用dHPLC进行HCV基因分型。
