Quantitative detection of hepatitis C virus RNA by polymerase chain reaction and dot blot hybridization

We have developed a new technique to quantify hepatitis C virus (HCV)-RNA using polymerase chain reaction (PCR) and dot blot hybridization. PCR was performed on 10-fold dilutions of an HCV-cDNA fragment for various numbers of cycles and on various serum volumes for 30 cycles. The resulting PCR products were spotted onto nitrocellulose filters and hybridized with a³²P-labelled internal probe. The intensity of the hybridization signal was proportional to the quantity of template cDNA and increased in proportion to the number of PCR cycles. Clear signals were detected after 30 cycles of PCR using an original serum volume of greater than 250 pl. Thus, this new technique allows the quantitation of HCV-RNA, and as it is simple and cost-effective, it promises to have many clinical applications.