For in vitro diagnostic use. Others cobas HBV Test 5800-6800-8800 IVD cobas® HBV RMD-58-68-88-HBV Quantitative nucleic acid test for use on the cobas® 5800/6800/8800 systems 09 040 820 190 9 040 820 190 09040820190 9040820190 09040820190 KIT COBAS 58/68/8800 HBV 192T IVD cobas HBV 00875197006414 Reagents, kits 1 kit 192 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. 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 run/batch control on the cobas® 5800/6800/8800 systems with the cobas® HBV, cobas® HCV, and cobas® HIV-1 tests en 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.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.The cobas® HBV is not intended for use as a screening test for the presence of HBV in blood or blood products or as a diagnostic test to confirm the presence of HBV infection. en cobas® HBV for use on the cobas® 5800/6800/8800 Systems 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.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. en cobas® HBV is based on fully automated sample preparation (nucleic acid extraction and purification) followed by PCR amplification and detection. The cobas® 5800 system is designed as one integrated instrument. The cobas® 6800/8800 systems consist of the sample supply module, the transfer module, the processing module, and the analytic module. Automated data management is performed by the cobas® 5800 or cobas® 6800/8800 system softwares 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 acid from patient samples, external controls and added lambda DNA (DNA-QS) molecules are simultaneously extracted. Viral nucleic acid is released by addition of proteinase and lysis reagent to the sample. The released nucleic acid binds to the silica surface of the added magnetic glass particles. Unbound substances and impurities, such as denatured protein, cellular debris and potential PCR inhibitors are removed with subsequent wash reagent steps and purified nucleic acid is eluted from the magnetic glass particles with elution buffer at elevated temperature. Selective amplification of target nucleic acid from the 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 enzyme is used for amplification. The master mix includes deoxyuridine triphosphate (dUTP), instead of deoxythimidine triphosphate (dTTP), which is incorporated into the newly synthesized DNA (amplicon). 14,17,18 Any contaminating amplicon from previous PCR runs are eliminated by the AmpErase enzyme, which is included in the PCR mix, during the first thermal cycling step. However, newly formed amplicon are not eliminated since the AmpErase enzyme is inactivated once exposed to temperatures above 55°C. 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 acts 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 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' 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 are possible. 12. Higuchi R, Dollinger G, Walsh PS, Griffith R. Simultaneous amplification and detection of specific DNA sequences. Biotechnology (N Y). 1992;10:413-7. PMID: 1368485. 13. Heid CA, Stevens J, Livak KJ, Williams PM. Real time quantitative PCR. Genome Res. 1996;6:986-94. PMID: 8908518. 14. Longo MC, Berninger MS, Hartley JL. Use of uracil DNA glycosylase to control carry-over contamination in polymerase chain reactions. Gene. 1990;93:125-8. PMID: 2227421. 17. Savva R, McAuley-Hecht K, Brown T, Pearl L. The structural basis of specific base-excision repair by uracil-DNA glycosylase. Nature. 1995;373:487-93. PMID: 7845459. 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;80:869-78. PMID: 7697717. en cobas® HBV is based on fully automated sample preparation (nucleic acid extraction and purification) followed by PCR amplification and detection. The cobas® 6800/8800 Systems consist of the sample supply module, the transfer module, the processing module, and the analytic module. Automated data management is performed by the cobas® 6800/8800 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 acid from patient samples, external controls and added lambda DNA (DNA-QS) molecules are simultaneously extracted.Viral nucleic acid is released by addition of proteinase and lysis reagent to the sample. The released nucleic acid binds to the silica surface of the added magnetic glass particles. Unbound substances and impurities, such as denatured protein, cellular debris and potential PCR inhibitors are removed with subsequent wash reagent steps and purified nucleic acid is eluted from the magnetic glass particles with elution buffer at elevated temperature.Selective amplification of target nucleic acid from the sample is achieved by the use of target virus-specific forward and reverse primers which are selected from highly conserved 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 enzyme is used for amplification. The master mix includes deoxyuridine triphosphate (dUTP), instead of deoxythimidine triphosphate (dTTP), which is incorporated into the newly synthesized DNA (amplicon).16,19,20 Any contaminating amplicon from previous PCR runs are eliminated by the AmpErase enzyme, which is included in the PCR mix, during the first thermal cycling step. However, newly formed amplicon are not eliminated since the AmpErase enzyme is inactivated once exposed to temperatures above 55°C.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 acts 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.14,15 When not bound to the target sequence, the fluorescent signal of the intact probe is 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' 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 are possible.14. Higuchi R, Dollinger G, Walsh PS, Griffith R. Simultaneous amplification and detection of specific DNA sequences. Biotechnology (N Y). 1992;10:413-7. PMID: 1368485.15. Heid CA, Stevens J, Livak KJ, Williams PM. Real time quantitative PCR. Genome Res. 1996;6:986-94. PMID: 8908518.16. Longo MC, Berninger MS, Hartley JL. Use of uracil DNA glycosylase to control carry-over contamination in polymerase chain reactions. Gene. 1990;93:125-8. PMID: 2227421.19. Savva R, McAuley-Hecht K, Brown T, Pearl L. The structural basis of specific base-excision repair by uracil-DNA glycosylase. Nature. 1995;373:487-93. PMID: 7845459.20. Mol CD, Arvai AS, Sanderson RJ, et al. Crystal structure of human uracil-DNA glycosylase in complex with a protein inhibitor: protein mimicry of DNA. Cell. 1995;82:701-8. PMID: 7671300. en cobas® HBV is based on fully automated sample preparation (nucleic acid extraction and purification) followed by PCR amplification and detection. The cobas® 6800/8800 Systems consist of the sample supply module, the transfer module, the processing module, and the analytic module. Automated data management is performed by the cobas® 6800/8800 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 acid from patient samples, external controls and added lambda DNA (DNA-QS) molecules are simultaneously extracted.Viral nucleic acid is released by addition of proteinase and lysis reagent to the sample. The released nucleic acid binds to the silica surface of the added magnetic glass particles. Unbound substances and impurities, such as denatured protein, cellular debris and potential PCR inhibitors are removed with subsequent wash reagent steps and purified nucleic acid is eluted from the magnetic glass particles with elution buffer at elevated temperature.Selective amplification of target nucleic acid from the sample is achieved by the use of target virus-specific forward and reverse primers which are selected from highly conserved 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 enzyme is used for amplification. The master mix includes deoxyuridine triphosphate (dUTP), instead of deoxythimidine triphosphate (dTTP), which is incorporated into the newly synthesized DNA (amplicon).14,17,18 Any contaminating amplicon from previous PCR runs are eliminated by the AmpErase enzyme, which is included in the PCR mix, during the first thermal cycling step. However, newly formed amplicon are not eliminated since the AmpErase enzyme is inactivated once exposed to temperatures above 55°C.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 acts 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 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' 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 are possible.12. Higuchi R, Dollinger G, Walsh PS, Griffith R. Simultaneous amplification and detection of specific DNA sequences. Biotechnology (N Y). 1992;10:413-7. PMID: 1368485.13. Heid CA, Stevens J, Livak KJ, Williams PM. Real time quantitative PCR. Genome Res. 1996;6:986-94. PMID: 8908518.14. Longo MC, Berninger MS, Hartley JL. Use of uracil DNA glycosylase to control carry-over contamination in polymerase chain reactions. Gene. 1990;93:125-8. PMID: 2227421.17. Savva R, McAuley-Hecht K, Brown T, Pearl L. The structural basis of specific base-excision repair by uracil-DNA glycosylase. Nature. 1995;373:487-93. PMID: 7845459.18. 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;80:869-78. PMID: 7697717 en

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