Journal of Hepatology 1994; 21:678-682 Printed hi Demnark . All rights reserved Munksgaard. Copenhagen Copyright© Journalof Hepatology1994 Journal of Hepatology ISSN 0168-8278 Rapid Publication Quantitation of HCV-replication using one-step competitive reverse transcription-polymerase chain reaction and a solid phase, colorimetric detection method Bernd Goergen l, Simone Jakobs I, Pete Symmons 2, Erik Hornes 2, Karl-Herrmann Meyer zum Btischenfelde I and Guido Gerken 1 JL Med. Klinik und Poliklinik. Johannes Gutenberg Universiffit Main=, Mainz, FRG and 2Dynal AS, Oslo, Norway (Received 8 February 1994) A solid phase assay for the colorimetric detection of competitively amplified HCV-cDNA has been established and used to investigate clinical samples from patients with chronic hepatitis. The assay is based on the reduction in the amplification of an hepatitis C virus-related competitor molecule by wild-type hepatitis C virus during polymerase chain reaction. The internal standard contains a lac operator sequence, allowing the amount of amplified competitor to be determined using a lac I-repressor/13-galactosidase fusion protein. The reduction in the amplification of competitor is dependent upon the concentration of HCV-RNA in the original sample. External hepatitis C virus wild-type standards are used to calibrate each concurrently tested set of patients. We present and discuss the potential benefit, but also the limitations of this new approach for quantifying hepatitis C virus viremia. In 47 serum samples from 28 patients with chronic hepatitis C virus infection, including five repeatedly tested aHCV positive patients under interferon therapy, viral titer was determined. Sera from nine healthy blood donors served as controls. The sensitivity and specificity of this procedure are identical to those of conventional nested pomymerase chain reaction. As both internal and external standards are used in every assay and final detection of amplicons can be carried out in microtiter plates, this reliable and time-saving test system may be routinely applied for monitoring antiviral treatment or for studying the relation of plus- and minus-stranded HCV-RNA in infected tissues. © Journal of Hepatology. Key words: Competitive reverse transcription-pomymerase chain reaction; Elisa; Hepatitis C; IFN-treatment; Lac oper- ator; Magnetic microspheres The development of polymerase chain reaction (PCR) has provided a highly sensitive diagnostic tool for the di- rect detection of HCV-RNA in serum or tissue specimens. Currently, reverse transcription (RT)-PCR is frequently used to identify low viremic hepatitis C virus (HCV) car- riers or to monitor antiviral treatment, and it thus com- plements serological assays detecting antibodies to several structural and non-structural viral antigens (for recent re- views, see (1, 2)). Nevertheless, the lack of standardized reaction protocols and a considerable risk of false positive results have hampered its routine application (3). Many attempts have been made to simplify and to quantify HCV RNA detection, for example by signal amplification assays or, limiting dilution PCR (4, 5). One of the most promising approaches for determining HCV levels in patient sera seems to be competitive PCR including varying amounts of the internal standard, but in its present format this technique is extremely time-con- suming and expensive (6). We have established a new and simpler HCV-RNA quantitation method, which is based upon the single coamplification of the patients' HCV- cDNA together with constant concentrations of the com- petitor-DNA and followed by a solid phase, colorimetric detection of PCR-products. Correspondence to: Guido Gerken, M.D., I. Med. Klinik und Poliklinik, Johannes Gutenberg Universit~itMainz, Langenbeckstr. I, 55101 Mainz, Federal Republic of Germany.