Automated quantitative determination of hepatitis C virus viremia by reverse transcription-PCR

Abstract

An automated reverse transcription-PCR was developed for the quantitative detection of hepatitis C virus. The quantitation is based on the coamplification and labelling with digoxigenin-dUTP during PCR of two similar templates, the viral genome and a modified RNA which acts as a mimic target. Known amounts of the mimic RNA sequence were introduced into the clinical samples. The automated quantitation of the two coamplified and labelled products depends on the use of two biotinylated caputre probes which are complementary, respectively, to a deleted sequence and to an inserted sequence introduced by site-directed mutagenesis in a wild viral cloned cDNA. This method proved to be simple, reproducible, and useful for quantitate hepatitis C virus viremia in chronically infected patients. This easy-to-perform, automated assay could also be used for the accurate determination of human immunodeficiency virus viremia or other RNA molecules.