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. 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.
Payan
C
C
Laboratoire de Virologie, Hôpital Broussais, Paris.
Dupré
T
T
Bélec
L
L
D001483:Base Sequence; D002908:Chronic Disease; D017931:DNA Primers; D016174:Hepacivirus / Q000302:isolation & purification*; D006526:Hepatitis C / Q000097:blood / Q000821:virology*; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012367:RNA, Viral / Q000032:analysis*; D012680:Sensitivity and Specificity; D013997:Time Factors; D014158:Transcription, Genetic
D017931:DNA Primers; D012367:RNA, Viral