DETECTION OF NEGATIVE-STRANDED HEPATITIS-C VIRUS-RNA USING A NOVEL STRAND-SPECIFIC REVERSE TRANSCRIPTION-POLYMERASE CHAIN-REACTION

We developed a novel single-tube reverse transcription-polymerase chai n reaction (RT-PCR) for the specific detection of negative-stranded he patitis C virus (HCV) RNA. By using in vitro synthesized positive-and negative-stranded HCV RNAs, it was demonstrated that as few as 50 copi es of negative-stranded HCV RNA could be specifically detected with a set of primers that amplify a 232-base pair sequence unique to the 5'- non-coding region of HCV RNA, while 10(8) copies of positive-stranded HCV RNA were not detected. In addition, we demonstrated that this meth od allows the detection as few as 100 copies of negative-stranded HCV RNA even with the coexistence of a 100-fold excess of positive-strande d HCV RNA. Furthermore, with this method, negative-stranded HCV RNA wa s detected in RNAs from liver biopsy specimens obtained from patients with chronic hepatitis C, but not in RNAs from HCV-positive sera. (C) 1998 Elsevier Science B.V. All rights reserved.