For in vitro diagnostic use. Others cobas HBV Test 4800 IVD cobas® HBV RMD-4800-HBV-001 Quantitative nucleic acid test for use on the cobas® 4800 system 06 979 564 190 6 979 564 190 06979564190 6979564190 06979564190 KIT COBAS 4800 HBV 120T CE-IVD cobas HBV 00875197005585 Reagents, kits 1 kit 120 tests true cobas® HBV: cobas® HBV is an in vitro nucleic acid amplification test for the quantitation of hepatitis B virus (HBV) DNA in human EDTA plasma or serum of HBV-infected individuals using the automated cobas® 4800 System for specimen processing, amplification and detection. This test is intended for use as an aid in the management of patients with chronic HBV infection undergoing anti-viral therapy. The test can be used to measure HBV DNA levels at baseline and during treatment to aid in assessing response to treatment. The results from cobas® HBV must be interpreted within the context of all relevant clinical and laboratory findings. 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® HBV 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 HBV DNA 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 lambda phage DNA 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 pre-core and core regions of HBV. Selective amplification of DNA QS is achieved by the use of sequence-specific forward and reverse primers which are selected to have no homology with the HBV genome. A thermostable DNA polymerase is used for PCR amplification. The master mix includes deoxyuridine triphosphate (dUTP), instead of deoxythymidine triphosphate (dTTP), which is incorporated into the newly synthesized DNA (amplicon).14,17,18 Any contaminating amplicons from previous PCR runs are inactivated as PCR templates by AmpErase, which is present in the master mix, prior to the first denaturation step of PCR. AmpErase catalyzes the removal of uracil from DNA, but has no activity on naturally occurring DNA, which does not contain uracil. Amplicon formed during subsequent cycles of PCR are not inactivated since AmpErase is inactive at the annealing and denaturation temperatures of PCR. The cobas® HBV master mix contains detection probes which are specific for the HBV target sequences and the QS nucleic acid, respectively. The specific HBV and DNA-QS detection probes are each labeled with one of two unique fluorescent dyes which act as a reporter. Each probe also has a second dye which acts as a quencher. The two reporter dyes are measured at defined wavelengths, thus permitting simultaneous detection and discrimination of the amplified HBV target and the DNA-QS.12,13 When not bound to the target sequence, the fluorescent signal of the intact probe is suppressed by the 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' exonuclease 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. Since the two specific reporter dyes are measured at defined wavelengths, simultaneous detection and discrimination of the amplified HBV target and the DNA-QS is possible. 12. 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. 13. Higuchi R, Dollinger G, Walsh PS, Griffith R. Simultaneous amplification and detection of specific DNA sequences. Bio/Technology. 1992;10:413-417. 14. Heid CA, Stevens J, Livak JK, Williams PM. Real time quantitative PCR. Genome Research. 1996; 6: 986-994. 17. 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. 18. 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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