COMPARISON OF A COMPETITIVE COMBINED REVERSE TRANSCRIPTION-PCR ASSAY WITH A BRANCHED-DNA ASSAY FOR HEPATITIS-C VIRUS-RNA QUANTITATION

We have developed a sensitive and reproducible one-step competitive re verse transcriptase (RT) PCR assay, which allows hepatitis C virus (HC V) RNA quantitation in plasma over a broad range of values, The RNA sa mples and a constant amount of an internal standard were reverse trans cribed and coamplified with the same primers in the same tube, A stand ard curve was obtained from an additional series of tubes containing b oth the internal standard and known amounts of a wild-type HCV RNA tra nscript, thus eliminating the need for titrating samples with the comp etitor, Eighty-eight anti-HCV-positive samples were tested by RT-PCR a nd a branched-DNA (bDNA) assay which has a detection limit of 3.5 x 10 (5) copies per ml, Fifty-five samples were quantifiable by both method s (correlation coefficient, 0.72), the ranges of values found by the R T-PCR and bDNA assays being, respectively, 0.127 x 10(6) to 18.4 x 10( 6) and 0.44 x 10(6) to 38 x 10(6) copies per ml, Six samples that had indeterminate values by the bDNA assay had RT-PCR values between 0.37 x 10(5) and 9.6 x 10(5) copies per ml, Twenty-two samples that had val ues below the cutoff value by the bDNA assay had RT-PCR values between 2.5 x 10(3) and 10.4 x 10(5) (18 less than and 4 more than the limit of 3.5 x 10(5) copies per ml), The remaining five samples were negativ e by both assays, The level of RT-PCR interassay reproducibility was h igh (correlation coefficient between duplicate values, 0.94), Our meth od, with a detection limit of 2,500 copies per ml, was markedly more s ensitive than the bDNA assay, This method is convenient for following up patients with low viremia, a common situation with alpha interferon treatment.