For in vitro diagnostic use. Others cobas HIV-1 Test 5800-6800-8800 IVD cobas® HIV-1 RMD-58-68-88-HIV1 Quantitative nucleic acid test for use on the cobas® 5800/6800/8800 systems 09 040 803 190 9 040 803 190 09040803190 9040803190 09040803190 KIT COBAS 58/68/8800 HIV 192T IVD cobas HIV-1 00875197006407 Reagents, kits 1 kit 192 tests true cobas® HIV-1 is an in vitro nucleic acid amplification test for the quantitation of human immunodeficiency virus type 1 (HIV-1) in EDTA plasma or from a cobas® Plasma Separation Card (PSC) dried plasma spot of HIV-1-infected individuals.This test is intended for use in conjunction with clinical presentation and other laboratory markers for the clinical management of HIV-1-infected patients. This test can be used for confirmation of HIV-1 infection in antibody reactive individuals and to assess patient prognosis by measuring the baseline HIV-1 level or to monitor the effects of antiretroviral therapy by measuring changes in HIV-1 RNA levels during the course of antiretroviral treatment. en cobas® HIV-1 is an in vitro nucleic acid amplification test for the quantitation of human immunodeficiency virus type 1 (HIV-1) in EDTA plasma of HIV-1-infected individuals using the automated cobas® 5800/6800/8800 Systems for specimen processing, amplification and detection. The test can quantitate HIV-1 RNA over the range of 20-10,000,000 cp/mL (33 to 1.67 x 107 International Units/mL).This test is intended for use in conjunction with clinical presentation and other laboratory markers for the clinical management of HIV-1 infected patients. The test can be used to assess patient prognosis by measuring the baseline HIV-1 level or to monitor the effects of antiretroviral therapy by measuring changes in HIV-1 RNA levels during the course of antiretroviral treatment.cobas® HIV-1 is not intended for use as a screening test for the presence of HIV-1 in donated blood or plasma or as a diagnostic test to confirm the presence of HIV-1 infection. en cobas® HIV-1 is an in vitro nucleic acid amplification test for the quantitation of human immunodeficiency virus type 1 (HIV-1) in EDTA plasma of HIV-1-infected individuals, using the automated cobas® 5800/6800/8800 Systems for specimen processing, amplification and detection. This test is intended for use in conjunction with clinical presentation and other laboratory markers for the clinical management of HIV-1-infected patients. This test can be used to assess patient prognosis by measuring the baseline HIV-1 level or to monitor the effects of antiretroviral therapy by measuring changes in HIV-1 RNA levels during the course of antiretroviral treatment.cobas® HIV-1 is not intended for use as a screening test for the presence of HIV-1 in donated blood or plasma or as a diagnostic test to confirm the presence of HIV-1 infection. en cobas® HIV-1 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 Systems software which assigns test results for all tests as target not detected, < LLoQ (lower limit of quantitation), > ULoQ (upper limit of quantitation) or HIV-1 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 acid from patient samples, external controls and added armored RNA (RNA-QS) molecules is simultaneously extracted. In summary, 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 the HIV-1 genome. The HIV-1 gag gene and the HIV-1 LTR region (dual target) are amplified by cobas® HIV-1. 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 HIV-1 genome. A thermostable DNA polymerase enzyme is used for both reverse-transcription and PCR amplification. The target and RNA-QS sequences are amplified simultaneously utilizing a PCR amplification profile with predefined temperature steps and number of cycles. The master mix includes deoxyuridine triphosphate (dUTP), instead of deoxythymidine triphosphate (dTTP), which is incorporated into the newly synthesized DNA (amplicon).5,6,7 Any contaminating amplicons from previous PCR runs are eliminated by the AmpErase enzyme, which is included in the PCR master mix, during the first thermal cycling step. However, newly formed amplicons are not eliminated since the AmpErase enzyme is inactivated once exposed to temperatures above 55°C.The cobas® HIV-1 master mix contains two detection probes specific for the HIV-1 target sequences and one for the RNA-QS. The probes are labeled with target specific fluorescent reporter dyes allowing simultaneous detection of HIV-1 target and RNA-QS in two different target channels.8,9 When not bound to the target sequence, the fluorescent signal of the intact probes 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. 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, respectively.5. 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: 2227421.6. 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: 7845459.7. 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: 7697717.8. Higuchi R, Dollinger G, Walsh PS, Griffith R. Simultaneous amplification and detection of specific DNA sequences. Biotechnology (N Y). 1992;10:413-7. PMID: 1368485.9. Heid CA, Stevens J, Livak KJ, Williams PM. Real time quantitative PCR. Genome Res. 1996;6:986-94. PMID: 8908518. en cobas® HIV-1 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 HIV-1 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 acid from patient samples, external controls and added armored RNA (RNA-QS) molecules is simultaneously extracted. In summary, 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 the HIV-1 genome. The HIV-1 gag gene and the HIV-1 LTR region (dual target) are amplified by cobas® HIV-1. 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 HIV-1 genome. A thermostable DNA polymerase enzyme is used for both reverse-transcription and PCR amplification. The target and RNA-QS sequences are amplified simultaneously utilizing a universal PCR amplification profile with predefined temperature steps and number of cycles. The master mix includes deoxyuridine triphosphate (dUTP), instead of deoxythimidine triphosphate (dTTP), which is incorporated into the newly synthesized DNA (amplicon).13,14,15 Any contaminating amplicon from previous PCR runs are eliminated by the AmpErase enzyme, which is included in the PCR master 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® HIV-1 master mix contains two detection probes specific for the HIV-1 target sequences and one for the RNA-QS. The probes are labeled with target specific fluorescent reporter dyes allowing simultaneous detection of HIV-1 target and RNA-QS in two different target channels.16,17 When not bound to the target sequence, the fluorescent signal of the intact probes 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. 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, respectively. 13. 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. 14. 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. 15. 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. 16. 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. 17. Heid CA, Stevens J, Livak KJ, Williams PM. Real time quantitative PCR. Genome Res. 1996;6:986-94. PMID: 8908518. en cobas® HIV-1 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 software which assigns test results for all tests as target not detected, < LLoQ (lower limit of quantitation), > ULoQ (upper limit of quantitation) or HIV-1 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 acid from patient samples, external controls and added armored RNA (RNA-QS) molecules is simultaneously extracted. In summary, 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 the HIV-1 genome. The HIV-1 gag gene and the HIV-1 LTR region (dual target) are amplified by cobas® HIV-1. 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 HIV-1 genome. A thermostable DNA polymerase enzyme is used for both reverse-transcription and PCR amplification. The target and RNA-QS sequences are amplified simultaneously utilizing a universal PCR amplification profile with predefined temperature steps and number of cycles. The master mix includes deoxyuridine triphosphate (dUTP), instead of deoxythimidine triphosphate (dTTP), which is incorporated into the newly synthesized DNA (amplicon).13,14,15 Any contaminating amplicon from previous PCR runs are eliminated by the AmpErase enzyme, which is included in the PCR master 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® HIV-1 master mix contains two detection probes specific for the HIV-1 target sequences and one for the RNA-QS. The probes are labeled with target specific fluorescent reporter dyes allowing simultaneous detection of HIV-1 target and RNA-QS in two different target channels.16,17 When not bound to the target sequence, the fluorescent signal of the intact probes 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. 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, respectively.13. Longo MC, Berninger MS, Hartley JL. Use of uracil DNA glycosylase to control carry-over contamination in polymerase chain reactions. Gene. 1990;93:125-128.14. Savva R, McAuley-Hecht K, Brown T, Pearl L. The structural basis of specific base-excision repair by uracil-DNA glycosylase. Nature. 1995;373:487-493.15. 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;80:869-878.16. Higuchi R, Dollinger G, Walsh PS, Griffith R. Simultaneous amplification and detection of specific DNA sequences. Biotechnology (NY). 1992;10:413-417.17. Heid CA, Stevens J, Livak JK, Williams PM. Real time quantitative PCR. Genome Res. 1996;6:986-994. en

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