We compared four primer sets from conserved regions of the hepatitis C virus (HCV) genome for their ability to detect HCV RNA in a "nested" cDNA polymerase chain reaction assay on sera from 114 anti-HCV antibodypositive individuals from around the world. The different primer sets had equivalent sensitivity, detecting <1 chimpanzee IDSO (dose that infects 50%) when tested against reference strain H of HCV. We tested equal amounts of RNA extracted from the serum of each individual with the four primer sets. The set derived from two highly conserved domains within the 5' noncoding (NC) region of the HCV genome, which also share significant similarity with Pestivirus 5' NC sequences, was the most effective at detecting HCV RNA. AU samples positive for HCV RNA with any other primer set were also positive with the primer set from the 5' NC region, and the latter was at least 3 times more likely to detect HCV infection than a primer set from within the nonstructural protein 3-like gene region (P < 0.001). We had no false positive results in >500 negative controls interspersed among the test samples. The 5' NC region primer set detected HCV-specific RNA, verified by highstringency Southern blot hybridization and DNA sequencing, in 100% of 15 acute and 33 chronic non-A, non-B hepatitis patients from the United States, Europe, and Asia, and 10 hepatocellular carcinoma patients from Africa and Asia that tested negative for the hepatitis B virus-encoded surface antigen. In conclusion, use of an appropriate primer set is crucial for detecting HCV RNA in the serum of infected individuals.Hepatitis C virus (HCV) is a positive-stranded RNA virus that appears to be the etiological agent of most posttransfusion non-A, non-B (NANB) hepatitis cases (1-3). Amplification of HCV RNA sequences by reverse transcription and cDNA polymerase chain reaction (cDNA PCR) is the only practical method currently available to demonstrate viremia in patients with HCV infection (3-14). However, genetic heterogeneity among different HCV strains (15-27) results in false-negative results in cDNA PCR assays because of primer and template mismatch. Therefore, to develop a reliable PCR assay for the diagnosis of HCV infection, we designed primers from conserved regions of the HCV genome (15,(28)(29)(30) and tested their ability to detect HCV RNA in a nested cDNA PCR assay in serum from 114 anti-HCV antibodypositive individuals from around the world. The results emphasize the importance of primer selection for the detection of HCV RNA.
MATERIALS AND METHODSWe tested serum samples from 114 individuals from 12 countries (Denmark,