Detection of hepatitis C virus RNA by a reliable, optimized single-step reverse transcription polymerase chain reaction.
Res Virol
; 146(5): 363-70, 1995.
Article
in En
| MEDLINE
| ID: mdl-8578010
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.
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Collection:
01-internacional
Database:
MEDLINE
Main subject:
RNA, Viral
/
Polymerase Chain Reaction
/
Hepatitis C
/
Hepacivirus
Type of study:
Diagnostic_studies
Limits:
Humans
Language:
En
Journal:
Res Virol
Journal subject:
VIROLOGIA
Year:
1995
Document type:
Article