Nested restriction site-specific PCR to detect and type hepatitis C virus (HCV): a rapid method to distinguish HCV subtype 1b from other genotypes

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 Ib 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,vith 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 Ib, 19 were predicted to contain subtype la, and 2 were predicted to contain subtype 3a. Among 54 samples predicted to contain HCV genotype Ib, all were confirmed by their 5' NC or NS5b sequences to be subtype Ib. Thus, both the sensitivity and specificity of the RSS-PCR test for the differentiation of HCV subtype Ib from the others were 100%. While the assay described here was designed to specifically differentiate HCV subtype Ib 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.