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

J Virol Methods. 1995 Jun;53(2-3):167-75. doi: 10.1016/0166-0934(95)00009-j.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acute Disease
  • Antibodies, Viral / blood
  • Base Sequence
  • Chronic Disease
  • DNA Primers
  • DNA, Viral / analysis
  • Hepacivirus / genetics
  • Hepacivirus / isolation & purification*
  • Hepatitis / blood
  • Hepatitis / virology*
  • Humans
  • Immunoenzyme Techniques
  • Molecular Sequence Data
  • Polymerase Chain Reaction / methods*
  • Prospective Studies
  • RNA, Viral / analysis
  • Sensitivity and Specificity
  • Transcription, Genetic

Substances

  • Antibodies, Viral
  • DNA Primers
  • DNA, Viral
  • RNA, Viral