In order to facilitate the characterizaton of HCV in plasma, we attempted to establish a quantitative competitive PCR (QC-PCR) for HCV RNA. For this purpose, we synthesized a mutant cDNA as a competitive template which shared the primers' sequences but differed from the wide type cDNA in having an internal deletion. Both mutant and wild type cDNAs contained 217 and 291 nucleotide sequences, respectively, from the 5' non-coding region of HCV. Both cDNAs were derived from a RNA extracted from a plasma sample by reverse transcription and DNA amplification with the same set of outer primers, and subseqently the first PCR product was amplified with inner primers within which either a mutant sense primer containing a sequence with an internal deletion of 74 nucleotides, or a wild type sense primer was used. Both cDNAs were cloned into the vector pCR II (TA-cloning kit, Invitrogen) and transformed in E. coli. The cloned HCV cDNA sequences were confirmed by DNA sequencing. Both wild type and mutant DNA plasmids were purified and quantitated by measuring absorbance at 260 nm. A known amount of the wild type plasmid was co- amplified with the serial diluted mutant plasmid solutions, and the resulting two bands corresponding to the expected sizes of the two plasmids can be demonstrated in a agarose gel. By measuring the incorporation of a labeled nucleotide or quantitating with a densitometer, we were able to obtain ratio of the two bands and found that the amount of wide type was indeed equivalent to that of the mutant plasmid when the ratio was 1. Thus, the number of HCV RNA molecules in an unknown sample can be calculated at the cDNA level if it is assumed that the efficiency of cDNA synthesis is 100%. We are in the process of applying this QA-PCR procedure to measure HCV RNA in plasma and its derived products. we also have synthesized RNAs from both wild type and mutant plasmids by Sp6 RNA polymeraseare in attempt to set up a similar QA-PCR procedure to quantitate the HCV RNA directly.
