NEW QUANTITATIVE ASSAY OF HEPATITIS-B AND HEPATITIS-C VIRUSES BY COMPETITIVE PCR USING ALTERNATIVE INTERNAL SEQUENCES

A competitive PCR was developed for quantitation of hepatitis B virus (HBV) DNA and hepatitis C virus (HCV) RNA, alternatively, using only t wo constructions containing both priming sites. DNAs corresponding to the HBV-S gene and the HCV-5' non-coding region were introduced into d istinct plasmids. HBV plasmid was used as a standard for HBV-DNA quant itation, in competition with the HCV plasmid as internal control. HBV and HCV plasmids also served as template for transcription of HBV RNA and HCV-RNA, which was used as internal control and standard, respecti vely, in competition for HCV-RNA quantitation. The analyzed samples fo r HBV and HCV quantitation were processed in the same way in competiti on with the internal controls and to the respective calibration curves obtained by serial dilutions of the mimic standard. This method showe d very good specificity and sensitivity, allowing absolute quantitatio n in a large linear range from 5 viral genomic copies per assay up to 10(6) copies, in sera of chronically HBV and HCV infected patients, as well as in supernatants of cell cultures inoculated with these viruse s. (C) 1997 Elsevier Science B.V.