Tang Yi-Wei, Li Haijing, Roberto Ann, Warner Diane, Yen-Lieberman Belinda
Department of Medicine, Vanderbilt University School of Medicine, Nashville, TN 37232, USA.
J Clin Virol. 2004 Oct;31(2):148-52. doi: 10.1016/j.jcv.2004.02.010.
Detection, quantitation and genotyping of hepatitis C virus (HCV) are important in selecting appropriate therapy. Current commercially available HCV genotyping kits, including sequencing-based TRUGENE HCV 5'NC and hybridization-based INNO-LiPA HCV II assays, rely on amplification products (amplicons) generated by HCV reverse-transcriptase (RT)-PCR methods such as the Roche AMPLICOR HCV test.
We developed a one-step RT-PCR assay to amplify and detect HCV RNA, and the resulting amplicons were used for HCV genotyping (TRUGENE). A total of 142 clinical samples were used to compare results from the RT-PCR/TRUGENE assay and those generated by the COBAS AMPLICOR and INNO-LiPA tests.
Eighty-seven of 108 plasma specimens which were positive by AMPLICOR were also positive by the user-developed RT-PCR, giving a sensitivity of 100.0%. The RT-PCR detected 2 of 21 AMPLICOR-negative specimens and none of 34 HCV-EIA-negative serum specimens, giving a specificity of 96.4%. The 87 amplicons from the RT-PCR yielded HCV genotypes. HCV genotype results from both TRUGENE and INNO-LiPA were all in agreement. The TRUGENE and INNO-LiPA assays identified 69 (79.3%) and 47 (54.0%) specimens, respectively at the subtype level. HCV subtype information agreed by both assays in 34 of 36 (99.4%) specimens. One specimen with HCV genotypes 2 and 4 by INNO-LiPA was classified as a single genotype 2 by TRUGENE.
Our data showed that the user-developed RT-PCR has comparable sensitivity and specificity for the detection of HCV in clinical specimens. The amplicons generated by the RT-PCR can be used for HCV genotyping by the sequencing-based TRUGENE assay.
丙型肝炎病毒（HCV）的检测、定量及基因分型对于选择合适的治疗方法很重要。目前市面上可买到的HCV基因分型试剂盒，包括基于测序的TRUGENE HCV 5'NC和基于杂交的INNO-LiPA HCV II检测法，都依赖于HCV逆转录酶（RT）-PCR方法（如罗氏AMPLICOR HCV检测法）产生的扩增产物（扩增子）。
我们开发了一种一步法RT-PCR检测法来扩增和检测HCV RNA，所得扩增子用于HCV基因分型（TRUGENE）。总共142份临床样本用于比较RT-PCR/TRUGENE检测法与COBAS AMPLICOR和INNO-LiPA检测法的结果。
108份经AMPLICOR检测呈阳性的血浆样本中有87份经自行开发的RT-PCR检测也呈阳性，灵敏度为100.0%。RT-PCR检测出21份AMPLICOR阴性样本中的2份，34份HCV-EIA阴性血清样本均未检测出，特异性为96.4%。RT-PCR的87个扩增子产生了HCV基因型。TRUGENE和INNO-LiPA的HCV基因型结果完全一致。TRUGENE和INNO-LiPA检测法分别在亚型水平上鉴定出69份（79.3%）和47份（54.0%）样本。两种检测法在36份样本中的34份（99.4%）中HCV亚型信息一致。一份经INNO-LiPA检测为HCV基因型2和4的样本经TRUGENE检测被归类为单一的基因型2。
我们的数据表明，自行开发的RT-PCR在检测临床样本中的HCV时具有相当的灵敏度和特异性。RT-PCR产生的扩增子可用于通过基于测序的TRUGENE检测法进行HCV基因分型。
