DETERMINATION OF HCV RNA CONCENTRATION BY DIRECT QUANTITATION OF THE PRODUCTS FROM A SINGLE RT-PCR

A novel method for the estimation of HCV RNA levels in vivo was develo ped, based on competitive RT-PCR. The use of the Tth DNA polymerase an d 5' P-32-labeled antisense primer respectively reduced cross-contamin ation and permitted the direct quantification of viral loads by the an alysis of the radioactivity of PCR products derived from a clinical sa mple and a competitive deleted template, separated previously on a pol yacrilamide gel. A HCV fragment (H) and a competitive (Delta H) RNA te mplates were synthesized for optimizing the method. The minimal starti ng RNA detectable by RT-PCR was approximate to 40 copies. RT-PCR perfo rmed with ratios Delta H/H ranging from 1/1 to 1/20 revealed different relative percentages of both H and Delta H products, changing from 90 % of Delta H product when the ratio was 1/1 to 5% when it was 1/20. Re gression analysis was adjusted to a linear model and served to further estimate HCV RNA loads from clinical samples. HCV RNA quantitation wa s carried out in 19 patients. Higher viral loads were related to type Ib infection and persistence of HCV RNA after interferon therapy. This method is simple, reproducible and useful for rapid estimation of HCV RNA load in vivo. (C) 1997 Elsevier Science B.V.