Development of a strand-specific RT-PCR based assay to detect the replicative form of hepatitis C virus RNA

The recent development of tagged RT-PCR and rTth RT-PCR has greatly improve d strand-specific detection of hepatitis C virus (HCV) RNA but these assays are still prone to some false detection of the incorrect strand of RNA. In this study we aimed to address additional factors which contribute towards false detection of HCV RNA. Firstly the benefits of both tagged primers an d the thermostable reverse transcriptase rTth during cDNA synthesis were co mbined and it was found that strand specificity was greatly improved withou t compromising sensitivity. The reliability of the assay was then optimised by addressing the following issues: control synthetic transcripts should b e free of contaminating plasmid DNA, residual RT activity should be minimis ed in the presence of PCR primers and cDNA should be free of unincorporated tagged RT primer prior to PCR amplification. The alterations made to the a ssay eliminated completely false detection of the incorrect strand of RNA i n the control assay whilst the correct strand was consistently detected at a cDNA dilution of 10(-3)-10(-4). Negative strand was not detected in RNA i solated from serum but was: detected, at a ten-fold lower level than positi ve strand, in RNA isolated from liver tissue. (C) 2001 Elsevier Science B.V . All rights reserved.