For in vitro diagnostic use. Others cobas HCV GT Test 4800 IVD cobas® HCV GT RMD-4800-HCVGT-001 HCV genotyping test for use on the cobas® 4800 system 06 984 274 190 6 984 274 190 06984274190 6984274190 06984274190 KIT C4800 HCV GT AMP/DET 120T CE-IVD cobas HCV GT 00875197005523 Reagents, kits 1 kit 120 tests true cobas® HCV GT is an in vitro nucleic acid amplification test for the identification of Hepatitis C Virus (HCV) genotypes 1 to 6 and genotype 1 subtypes a and b in human plasma or serum from individuals with chronic HCV infection, using the cobas® 4800 system, the cobas® x 480 Instrument for automated specimen processing and the cobas® z 480 Analyzer for automated amplification and detection. The cobas® HCV GT assists healthcare providers in managing patients with previously confirmed chronic HCV infection by identifying specific HCV genotypes and subtypes. cobas® HCV GT is not intended for use as a screening test for the presence of HCV in blood or blood products, or as a diagnostic test to confirm the presence of HCV infection. en cobas® HCV GT is an in vitro nucleic acid amplification test for the qualitative identification of Hepatitis C Virus (HCV) genotypes 1 to 6 and genotype 1 subtypes a and b in human EDTA plasma or serum from individuals with chronic HCV infection, using the cobas® 4800 system: the cobas® x 480 instrument for automated sample processing and the cobas® z 480 analyzer for automated amplification and detection. cobas® HCV GT is intended for use in selecting individuals with chronic HCV infection for antiviral therapy and in determining the duration of therapy regimens according to the antiviral therapy prescribing information. en cobas® HCV GT is based on fully automated sample preparation (nucleic acid extraction and purification) followed by real-time RT-PCR amplification and detection. The cobas® 4800 System consists of the cobas® x 480 instrument for automated sample processing and the cobas® z 480 analyzer for automated amplification and detection. Automated data management is performed by the cobas® 4800 software which assigns test results for all tests as one or more genotypes and subtypes, Indeterminate (HCV RNA detected but no genotype or subtype identified) or Invalid (HCV RNA not detected). Results can be reviewed directly on the system screen, exported, or printed as a report. Nucleic acids from patient samples and external controls are released by addition of proteinase and a chaotropic lysis reagent to the sample. The released nucleic acids bind to the silica surface of magnetic glass particles. Unbound substances and impurities, such as denatured proteins, cellular debris and potential PCR inhibitors, are removed with subsequent wash steps, and purified nucleic acids are eluted from the magnetic glass particles with elution buffer at elevated temperature. Each sample is amplified in three real-time RT-PCR reactions. Genotype and subtype specific RT-PCR amplification and detection of HCV genotypes 1 to 6 and subtypes 1a and 1b is achieved through the use of genotype and subtype specific primers and fluorescent dye-labeled probes. Each reaction also includes primers and probes for a highly conserved region of the HCV genome, for amplification and detection of HCV regardless of genotype, as an internal control. The probes are labeled with four different fluorescent reporter dyes, allowing simultaneous detection of HCV and up to three genotypes or subtypes in each reaction. 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).7,8,9 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. Each of the oligonucleotide probes in the cobas® HCV GT master mixes is labeled with a non-fluorescent quencher dye and a fluorescent reporter dye. When probes are intact the fluorescence of the reporter dyes is suppressed by the quencher dye. During PCR amplification, the probes hybridize to their target sites between the primer binding sites, and DNA polymerase extends the primers. The 5'-to-3' nuclease activity of the DNA polymerase cleaves the hybridized probes, 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. Real-time detection and discrimination of PCR products is accomplished by measuring the fluorescence of the released reporter dyes in each cycle of PCR, which is done automatically by the cobas® z 480 analyzer. 7. 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. 8. 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. 9. 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. en

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