Single-step reverse transcription-polymerase chain reaction for hepatitis C virus RNA with DNA enzyme immunoassay...

A single reverse transcription-polymerase chain reaction (SRT-PCR) for HCV RNA detection, confirmed by hybridization of amplified products with biotinylated probes using DNA enzyme immunoassay (DEIA), was compared to nested-PCR (N-PCR) on 20 sera from patients with chronic (n = 18) or acute (n = 2) hepatitis. Results obtained by SRT-PCR confirmed by DEIA and by N-PCR identical. All but one of the patients with chronic hepatitis and positive HCV serology as well as the patients with acute hepatitis had detectable HCV RNA in serum; all patients with chronic hepatitis and indeterminate HCV serology and all controls (n = 5) were negative by the two PCR methods. Both SRT-PCR and N-PCR remained positive after 7 x 10(-2) and 5 x 10(-4) dilutions of two HCV RNA-positive sera. The threshold of detection for SRT-PCR was 15 RNA copies per assay, as assessed by testing serial dilutions of an in vitro synthesized 5'-NCR HCV RNA transcript. SRT-PCR confirmed by DEIA for HCV RNA appears to be as sensitive and specific as N-PCR. Furthermore, it is easier to perform, with less of contamination, is less time-consuming, requires fewer enzymes, and it permits semi-quantification of PCR products.