QUANTIFICATION OF HEPATITIS-C VIRUS-RNA BY COMPETITIVE AMPLIFICATION OF RNA FROM DENATURED SERUM AND HYBRIDIZATION ON MICROTITER PLATES

The direct detection of hepatitis C virus (HCV) RNA by PCR is widely u sed to determine the presence of circulating virions. The most relevan t limit of this approach is the lack of quantitative information about the viral titer. We report a technique of competitive amplification a llowing the estimation of HCV RNA copy number in biological samples. W e constructed a standard competitive RNA template containing only two point mutations compared,vith its wild-type counterpart. The competito r was added in titrated amounts to the target RNA, and the mixture was then reverse transcribed and amplified in the same reaction tube. The relative amounts of target and competitor were determined by differen tial hybridization on microtiter plates with nonradioactive probes. Th e evaluation of HCV RNA titer required a single coamplification with t he competitor and could be read from a standard curve. Furthermore, th is method proved suitable for amplification of HCV RNA directly from s erum, thus avoiding the intrinsic variability of the RNA extraction st ep.