QUANTITATION OF HCV-REPLICATION USING ONE-STEP COMPETITIVE REVERSE TRANSCRIPTION-POLYMERASE CHAIN-REACTION AND A SOLID-PHASE, COLORIMETRIC DETECTION METHOD

A solid phase assay for the colorimetric detection of competitively am plified HCV-cDNA has been established and used to investigate clinical samples from patients with chronic hepatitis. The assay is based on t he reduction in the amplification of an hepatitis C virus-related comp etitor molecule by wild-type hepatitis C virus during polymerase chain reaction. The internal standard contains a lac operator sequence, all owing the amount of amplified competitor to be determined using a lac I-repressor/beta-galactosidase fusion protein. The reduction in the am plification of competitor is dependent upon the concentration of HCV-R NA in the original sample. External hepatitis C virus wild-type standa rds are used to calibrate each concurrently tested set of patients. We present and discuss the potential benefit, but also the limitations o f this new approach for quantifying hepatitis C virus viremia. In 47 s erum samples from 28 patients with chronic hepatitis C virus infection , including five repeatedly tested alpha HCV positive patients under i nterferon therapy, viral titer was determined. Sera from nine healthy blood donors served as controls. The sensitivity and specificity of th is procedure are identical to those of conventional nested pomymerase chain reaction. As both internal and external standards are used in ev ery assay and final detection of amplicons can be carried out in micro titer plates, this reliable and time-saving test system may be routine ly applied for monitoring antiviral treatment or for studying the rela tion of plus- and minus-stranded HCV-RNA in infected tissues. (C) Jour nal of Hepatology.