HEPATITIS-C VIRUS DETECTION BY SINGLE-ROUND PCR SPECIFIC FOR THE TERMINAL 3'-NONCODING REGION

A single-round PCR method with primers specific for the 3' noncoding r egion (NCR) of hepatitis C virus (HCV) has been developed, Using a dou ble RNAzol-B extraction, a high-temperature reverse-transcription step with SuperScript II reverse transcriptase, and a 40-cycle two-tempera ture PCR with a TaqStart antibody hot-start procedure, we were able to detect a 92-nucleotide fragment of the recently discovered 98-nucleot ide highly conserved sequence at the 3' terminus of the HCV genome, Di rect sequencing of the PCR products confirmed the specificity of the P CR and demonstrated conservation in this region, Only one nucleotide c hange in 14 specimens was found, End point dilution titration of sera with known viral RNA titers showed the sensitivity of the single-round 3' NCR PCR to he comparable to those of the established nested 5' NCR assays (fewer than 25 HCV genome equivalents),To evaluate specificity and sensitivity, a panel of 116 serum samples characterized by nested 5'-end PCR, genotyping, and quantitative assays was tested, A high de gree of concordance (96%) between the 3' NCR and 5' NCR PCR results wa s found, The sequence conservation at the 3' end of the HCV genome amo ng common genotypes and the savings in time, labor, and reagents from a single-round PCR make this assay a useful addition to the detection systems available to identify and monitor HCV infection.