Importance of primer selection for the detection of hepatitis C virus RNA with the polymerase chain reaction assay.

Abstract

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,

Long-term mortality and morbidity of transfusion-associated non-A, non-B, and type C hepatitis: A National Heart, Lung, and Blood Institute collaborative study

“…PCR-based amplification was done with primers homologous to sequences in the 5Ј-noncoding region, and colorometric detection of PCR products was performed using a commercial kit. 13,14 HCV-RNA quality control materials were prepared from a sample of HCV (strain H) with a concentration of 10 6.5 CID 50 per mL (gift of Dr. Robert Purcell, Hepatitis Viruses Section, NIAID). Assays of samples containing 300 CID 50 per mL (about 5400 HCV RNA copies per mL)…”

Variation of the hepatitis C virus 5' non-coding region: implications for secondary structure, virus detection and typing

“…The 5'NCR is often used as the target for detection of HCV RNA by RT-PCR amplification and there is evidence that the efficiency of virus RNA detection is higher for the 5'NCR than for other regions of the genome (Bukh et al, 1992b;Castillo et al, 1992;Xu et al, 1994). However, as documented above, genotypespecific sequence polymorphisms exist throughout the 5'NCR except for a highly conserved 60 nucleotide region at the 3' end.…”

Classification of hepatitis C virus into six major genotypes and a series of subtypes by phylogenetic analysis of the NS-5 region

“…encoding the putative envelope protiens [El, E2/nonstructural protein 1 (NS-1)] are the most variable Hijikata et al, 1991), whereas the 5' non-coding region (5' NCR) is the most conserved Cha et al, 1991 ;Okamoto et al, 1990;Bukh et al, 1992a). Comparison of published sequences of HCV has led to the identification of a number of distinct virus' types', that may differ from each other by as much as 33 % over the whole viral genome (Choo et aI., 1991 ;Okamoto et al, 1991;Chan et al, 1992;Mori et al, 1992;Okamoto et al, 1992b).…”