DETECTION OF HEPATITIS-C VIRUS-RNA BY A RELIABLE, OPTIMIZED SINGLE-STEP REVERSE TRANSCRIPTION-POLYMERASE CHAIN-REACTION

A single-step reverse transcription polymerase chain reaction (SRT-PCR ) method was optimized for hepatitis C virus (HCV) RNA detection. Extr action procedures by proteinase K and guanidinium isothiocyanate gave similar results. The optimal MgCl2 concentration for the SRT-PCR metho d was 2 mM with 10 units of superscript M-MLV RNase H- reverse transcr iptase and 1 unit of Taq polymerase. Shorter PCR cycling steps gave a 10-fold-increased PCR product compared with longer cycling steps. Twen ty-five anti-HCV-positive sera from chronic hepatitis C patients were positive with SRT-PCR, whereas only 17 out of 25 were positive by diss ociated RT and PCR (dRT/PCR). Specificity was assessed by twenty negat ive 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.