Detection of hepatitis C virus RNA by a reliable, optimized single-step reverse transcription polymerase chain reaction

Res Virol. Sep-Oct 1995;146(5):363-70. doi: 10.1016/0923-2516(96)80599-1.

Abstract

A single-step reverse transcription polymerase chain reaction (SRT-PCR) method was optimized for hepatitis C virus (HCV) RNA detection. Extraction procedures by proteinase K and guanidinium isothiocyanate gave similar results. The optimal MgCl2 concentration for the SRT-PCR method was 2 mM with 10 units of superscript M-MLV RNase H-reverse transcriptase and 1 unit of Taq polymerase. Shorter PCR cycling steps gave a 10-fold-increased PCR product compared with longer cycling steps. Twenty-five anti-HCV-positive sera from chronic hepatitis C patients were positive with SRT-PCR, whereas only 17 out of 25 were positive by dissociated RT and PCR (dRT/PCR). Specificity was assessed by twenty negative controls. SRT-PCR was 5-fold more sensitive (5 HCV RNA copies per assay) than dRT/PCR with an HCV RNA transcript. Our SRT-PCR method for HCV RNA detection appears fully adapted for routine use in a medical virology laboratory.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Sequence
  • Chronic Disease
  • DNA Primers
  • Hepacivirus / isolation & purification*
  • Hepatitis C / blood
  • Hepatitis C / virology*
  • Humans
  • Molecular Sequence Data
  • Polymerase Chain Reaction / methods*
  • RNA, Viral / analysis*
  • Sensitivity and Specificity
  • Time Factors
  • Transcription, Genetic

Substances

  • DNA Primers
  • RNA, Viral