Strand-Specific rTth RT-PCR for the Analysis of HCV Replication

Abstract

Because of the very low level of HCV present in the serum of infected individuals, as well as the low level of replication in the host, reverse transcription-polymerase chain reaction (RT-PCR) assays are the only method suitable for the routine detection of HCV RNA. The use of RT-PCR to monitor HCV replication in vivo as well as the in vitro inoculation of cultured cells presents unique problems. The extreme sensitivity of PCR permits the detection of HCV RNA in tissues not permissive for replication of the virus, and following in vitro infections, the residual inoculum can be detected for extended time periods depending on the sensitivity of the PCR procedure. Since HCV is a positive-stranded RNA virus, the detection of negative-strand RNA should be indicative of active viral RNA replication, assuming that the inoculum contains primarily positive-strand RNA. Early attempts to detect negative-strand RNA employed a strand-specific PCR technique that utilized only one primer during cDNA synthesis, followed by inactivation of the RT and amplification of the cDNA by PCR. During the course of our studies, we found this technique to lack significant strand specificity using synthetic RNA. A series of experiments suggested that the lack of specificity was probably owing to a combination of factors, including false priming of the incorrect strand (e.g., the positive strand in a negative-strand assay) by the cDNA primer, self-priming of the RNA, and random priming by extraneous nucleic acids (illustrated in Fig. 1 A).