Comparison of 5' noncoding-core with 5' noncoding regions of HCV by RT-PCR: importance and clinical implications

The molecular detection of HCV RNA by using primers from the 5' noncoding region (5'NCR) is universally accepted and preferred for diagnosis; however, insufficient sequence variation limits its usefulness for differentiating various subtypes-such as 1a/1b and newer subtypes, such as 1c-which cannot be distinguished by analysis of this region alone. Determination of genotypes and subtypes has important implications in the clinical management and epidemiologic investigations. The present study was designed to develop a reverse transcription-polymerase chain reaction (RT-PCR) assay using degenerate primers from the 5' noncoding-core region (5'NCR-core region) for detecting as well as subtyping HCV isolates. 5'NCR-core region and 5'NCR were amplified by nested PCR. Genotyping of HCV isolates was carried out using restriction fragment length polymorphism (RFLP) assay from the 5'NCR-core region using restriction enzymes AccI, MboI, and BstNI. The accuracy of the RFLP method was also evaluated using direct sequencing.