For in vitro diagnostic use. Others cobas HCV Test 4800 IVD cobas® HCV RMD-4800-HCV-001 Quantitative nucleic acid test for use on the cobas® 4800 system 06 979 602 190 6 979 602 190 06979602190 6979602190 06979602190 KIT COBAS 4800 HCV 120T CE-IVD cobas HCV 00875197005530 Reagents, kits 1 kit 120 tests true cobas® HCV: cobas® HCV is an in vitro nucleic acid amplification test for both the detection and quantitation of hepatitis C virus (HCV) RNA, in human EDTA plasma or serum, of HCV-infected individuals. Samples containing HCV genotype 1 to 6 are validated for detection and quantitation in the assay. The test is intended for use as an aid in the diagnosis of HCV infection in the following populations: individuals with antibody evidence of HCV with evidence of liver disease, individuals suspected to be actively infected with HCV antibody evidence, and individuals at risk for HCV infection with antibodies to HCV. Detection of HCV RNA indicates that the virus is replicating and therefore is evidence of active infection. The test is intended for use as an aid in the management of HCV-infected patients undergoing anti-viral therapy. The assay measures HCV RNA levels at baseline and during treatment and can be utilized to predict sustained and non-sustained virological response to HCV therapy. The results must be interpreted within the context of all relevant clinical and laboratory finding. cobas® HBV/HCV/HIV-1 Control Kit: cobas® HBV/HCV/HIV-1 Control Kit is intended for use as a positive and negative run control on the cobas® 4800 System with the cobas® HBV, cobas® HCV, and cobas® HIV-1 tests. en cobas® HCV is an in vitro nucleic acid amplification test for both the detection and quantitation of hepatitis C virus (HCV) RNA, in human EDTA plasma or serum, of HCV antibody positive or HCV-infected individuals. Specimens containing HCV genotypes 1 to 6 are validated for detection and quantitation in the assay. cobas® HCV is intended for use as an aid in the diagnosis of HCV infection in the following populations: individuals with antibody evidence of HCV with evidence of liver disease, individuals suspected to be actively infected with HCV antibody evidence, and individuals at risk for HCV infection with antibodies to HCV. Detection of HCV RNA indicates that the virus is replicating and therefore is evidence of active infection. cobas® HCV is intended for use as an aid in the management of HCV-infected patients undergoing anti-viral therapy. The assay can be used to measure HCV RNA levels at baseline, during treatment, at the end of treatment, and at the end of follow up of treatment to determine sustained or non-sustained viral response. The results must be interpreted within the context of all relevant clinical and laboratory findings. cobas® HCV has not been approved for use as a screening test for the presence of HCV in blood or blood products. Assay performance characteristics have been established for individuals treated with certain direct-acting antiviral agents (DAA) regimens. No information is available on the assay’s predictive value when other DAA combination therapies are used. en cobas® HCV is based on fully automated sample preparation (nucleic acid extraction and purification) followed by PCR amplification and detection. The cobas® 4800 System consists of the cobas x 480 instrument and the cobas z 480 analyzer. Automated data management is performed by the cobas® 4800 software which assigns test results for all tests as target not detected, < LLoQ (lower limit of quantitation), > ULoQ (upper limit of quantitation) or HCV RNA detected, a value in the linear range LLoQ ≤ x ≤ ULoQ. Results can be reviewed directly on the system screen, exported, or printed as a report. Nucleic acids from patient samples, external controls and added armored RNA QS molecules are simultaneously extracted. In summary, viral nucleic acids are released by addition of proteinase and lysis reagent to the sample. The released nucleic acids bind to the silica surface of the added magnetic glass particles. Unbound substances and impurities, such as denatured proteins, cellular debris and potential PCR inhibitors are removed with subsequent wash buffer steps and purified nucleic acids are eluted from the magnetic glass particles with elution buffer at elevated temperature. Selective amplification of target nucleic acids from the patient sample is achieved by the use of target virus-specific forward and reverse primers which are selected from highly conserved regions of HCV. Selective amplification of RNA QS is achieved by the use of sequence-specific forward and reverse primers which are selected to have no homology with the HCV genome. A thermostable DNA polymerase is used for both reverse-transcription and PCR amplification. The master mixes include deoxyuridine triphosphate (dUTP), instead of deoxythymidine triphosphate (dTTP), which is incorporated into the newly synthesized DNA (amplicon).22,23,24 Any contaminating amplicons from previous PCR runs are inactivated as PCR templates by AmpErase, which is present in the master mixes, prior to the first denaturation step of PCR. AmpErase catalyzes the removal of uracil from DNA, but has no activity on RNA or naturally occurring DNA, which does not contain uracil. Amplicons formed during subsequent cycles of PCR are not inactivated since AmpErase is inactive at the annealing and denaturation temperatures of PCR. cobas® HCV master mix contains dual detection probes specific for the HCV target sequences and one detection probe for the RNA QS. The probes are labeled with target-specific fluorescent reporter dyes allowing simultaneous detection of HCV target and RNA QS in two different detection channels.25,26 When not bound to the target sequence, the fluorescent signals of the intact probes are suppressed by a quencher dye. During the PCR amplification step, hybridization of the probes to the specific single-stranded DNA template results in cleavage of the probe by the 5'-to-3' nuclease activity of the DNA polymerase resulting in separation of the reporter and quencher dyes and the generation of a fluorescent signal. With each PCR cycle, increasing amounts of cleaved probes are generated and the cumulative signal of the reporter dye increases concomitantly. Real-time detection and discrimination of PCR products is accomplished by measuring the fluorescence of the released reporter dyes for the viral targets and RNA QS. 22. Longo MC, Berninger MS, Hartley JL. Use of uracil DNA glycosylase to control carry-over contamination in polymerase chain reactions. Gene. 1990;93:125-128. 23. Savva R, McAuley-Hecht K, Brown T, Pearl L. The structural basis of specific base-excision repair by uracil-DNA glycosylase. Nature. 1995;373:487-493. 24. Mol CD, Arvai AS, Slupphaug G, et al. Crystal structure and mutational analysis of human uracil-DNA glycosylase: structural basis for specificity and catalysis. Cell. 1995;80:869-878. 25. Higuchi R, Dollinger G, Walsh PS, Griffith R. Simultaneous amplification and detection of specific DNA sequences. Biotechnology (NY). 1992;10:413-417. 26. Heid CA, Stevens J, Livak JK, Williams PM. Real time quantitative PCR. Genome Res. 1996;6:986-994. en cobas® HCV is based on fully automated sample preparation (nucleic acid extraction and purification) followed by PCR amplification and detection. The cobas® 4800 System consists of the cobas x 480 instrument and the cobas z 480 analyzer. Automated data management is performed by the cobas® 4800 software which assigns test results for all tests as target not detected, < LLoQ (lower limit of quantitation), > ULoQ (upper limit of quantitation) or HCV RNA detected, a value in the linear range LLoQ ≤ x ≤ ULoQ. Results can be reviewed directly on the system screen, exported, or printed as a report. Nucleic acids from patient samples, external controls and added armored RNA QS molecules are simultaneously extracted. In summary, viral nucleic acids are released by addition of proteinase and lysis reagent to the sample. The released nucleic acids bind to the silica surface of the added magnetic glass particles. Unbound substances and impurities, such as denatured proteins, cellular debris and potential PCR inhibitors are removed with subsequent wash buffer steps and purified nucleic acids are eluted from the magnetic glass particles with elution buffer at elevated temperature. Selective amplification of target nucleic acids from the patient sample is achieved by the use of target virus-specific forward and reverse primers which are selected from the highly conserved untranslated region (5'-UTR) of the HCV genome. Selective amplification of RNA QS is achieved by the use of sequence-specific forward and reverse primers which are selected to have no homology with the HCV genome. A thermostable DNA polymerase is used for both reverse-transcription and PCR amplification. The master mixes include deoxyuridine triphosphate (dUTP), instead of deoxythymidine triphosphate (dTTP), which is incorporated into the newly synthesized DNA (amplicon).22,23,24 Any contaminating amplicons from previous PCR runs are inactivated as PCR templates by AmpErase, which is present in the master mixes, prior to the first denaturation step of PCR. AmpErase catalyzes the removal of uracil from DNA, but has no activity on RNA or naturally occurring DNA, which does not contain uracil. Amplicons formed during subsequent cycles of PCR are not inactivated since AmpErase is inactive at the annealing and denaturation temperatures of PCR. cobas® HCV master mix contains dual detection probes specific for the HCV target sequences and one detection probe for the RNA QS. The probes are labeled with target-specific fluorescent reporter dyes allowing simultaneous detection of HCV target and RNA QS in two different detection channels.25,26 When not bound to the target sequence, the fluorescent signals of the intact probes are suppressed by a quencher dye. During the PCR amplification step, hybridization of the probes to the specific single-stranded DNA template results in cleavage of the probe by the 5'-to-3' nuclease activity of the DNA polymerase resulting in separation of the reporter and quencher dyes and the generation of a fluorescent signal. With each PCR cycle, increasing amounts of cleaved probes are generated and the cumulative signal of the reporter dye increases concomitantly. Real-time detection and discrimination of PCR products is accomplished by measuring the fluorescence of the released reporter dyes for the viral targets and RNA QS. 22. Longo MC, Berninger MS, Hartley JL. Use of uracil DNA glycosylase to control carry-over contamination in polymerase chain reactions. Gene. 1990;93:125-128. 23. Savva R, McAuley-Hecht K, Brown T, Pearl L. The structural basis of specific base-excision repair by uracil-DNA glycosylase. Nature. 1995;373:487-493. 24. Mol CD, Arvai AS, Slupphaug G, et al. Crystal structure and mutational analysis of human uracil-DNA glycosylase: structural basis for specificity and catalysis. Cell. 1995;80:869-878. 25. Higuchi R, Dollinger G, Walsh PS, Griffith R. Simultaneous amplification and detection of specific DNA sequences. Biotechnology (NY). 1992;10:413-417. 26. Heid CA, Stevens J, Livak JK, Williams PM. Real time quantitative PCR. Genome Res. 1996;6:986-994. en

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