Krekulova L, Rehak V, Wakil A E, Harris E, Riley L W
Division of Infectious Diseases, School of Public Health, University of California-Berkeley, Berkeley, CA 94720, USA.
J Clin Microbiol. 2001 May;39(5):1774-80. doi: 10.1128/JCM.39.5.1774-1780.2001.
Genotypic differentiation of hepatitis C virus (HCV) has become an integral part of clinical management and epidemiologic studies of hepatitis C infections. Thus, it is extremely important in areas such as the Czech Republic, where current instrumentation and kits for assessing HCV infection are too costly for widespread use. We describe a new and relatively inexpensive method called nested restriction site-specific PCR (RSS-PCR) that generates a "fingerprint" pattern to represent an HCV genotype without the use of restriction endonucleases and that specifically differentiates HCV genotype 1b from the other HCV genotypes. The RSS-PCR method was applied directly to serum samples from patients with hepatitis C from the Czech Republic and from patients with known HCV genotypes from the United States. The method was validated by comparison of the subtype determined by RSS-PCR to the subtype determined from analysis of the 5' noncoding region (NC) or the nonstructural protein gene (NS5b) nucleotide sequence of HCV in these clinical samples. From 75 Czech samples containing HCV RNA, three distinct RSS-PCR patterns were observed; 54 were predicted to contain subtype 1b, 19 were predicted to contain subtype 1a, and 2 were predicted to contain subtype 3a. Among 54 samples predicted to contain HCV genotype 1b, all were confirmed by their 5' NC or NS5b sequences to be subtype 1b. Thus, both the sensitivity and specificity of the RSS-PCR test for the differentiation of HCV subtype 1b from the others were 100%. While the assay described here was designed to specifically differentiate HCV subtype 1b from the other HCV genotypes, the RSS-PCR method can be modified to differentiate any HCV genotype or subtype of interest. Its simplicity and speed may provide new opportunities to study the epidemiology of HCV infections and the relationship between HCV genotypes and clinical outcome by more laboratories throughout the world.
丙型肝炎病毒（HCV）的基因分型已成为丙型肝炎感染临床管理和流行病学研究不可或缺的一部分。因此，在捷克共和国这样的地区，这一点极为重要，因为目前用于评估HCV感染的仪器和试剂盒成本过高，无法广泛使用。我们描述了一种新的且相对便宜的方法，称为巢式限制性位点特异性PCR（RSS-PCR），该方法无需使用限制性内切酶即可生成代表HCV基因型的“指纹”图谱，并能特异性地将HCV 1b基因型与其他HCV基因型区分开来。RSS-PCR方法直接应用于来自捷克共和国丙型肝炎患者以及来自美国已知HCV基因型患者的血清样本。通过将RSS-PCR确定的亚型与这些临床样本中HCV的5'非编码区（NC）或非结构蛋白基因（NS5b）核苷酸序列分析确定的亚型进行比较，对该方法进行了验证。从75份含有HCV RNA的捷克样本中，观察到三种不同的RSS-PCR图谱；预计54份含有1b亚型，19份含有1a亚型，2份含有3a亚型。在预计含有HCV 1b基因型的54份样本中，所有样本经其5' NC或NS5b序列确认均为1b亚型。因此，RSS-PCR检测区分HCV 1b亚型与其他亚型的敏感性和特异性均为100%。虽然这里描述的检测方法旨在特异性地将HCV 1b亚型与其他HCV基因型区分开来，但RSS-PCR方法可以进行修改，以区分任何感兴趣的HCV基因型或亚型。其简单性和速度可能为世界各地更多实验室研究HCV感染的流行病学以及HCV基因型与临床结果之间的关系提供新的机会。
