For in vitro diagnostic use. Others cobas HIV-1 Test 4800 IVD cobas® HIV-1 Test RMD-4800-HIV1-002 Nucleic acid test for use on the cobas® 4800 system 08 792 992 190 8 792 992 190 08792992190 8792992190 08792992190 KIT COBAS 4800 HIV-1 QT-QL 120T CE-IVD cobas HIV-1 Test 00875197006209 Reagents, kits 1 kit 120 tests true cobas® HIV-1: 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 and for the qualitative detection of HIV-1 in dried blood spots of HIV-1-infected infants born to mothers infected with HIV-1. The test is to be used with cobas® 4800 System.This test is intended for use in conjunction with clinical presentation and other laboratory markers of disease progression for the clinical management of HIV-1-infected patients. When used as a quantitative test, this test can be used as an aid in diagnosis for confirmation of HIV-1 infection in antibody reactive individuals and to assess patient prognosis by measuring the baseline HIV-1 RNA level or to monitor the effects of antiretroviral therapy by measuring changes in HIV-1 RNA levels during the course of antiretroviral treatment. When used as a qualitative test with dried blood spots, detection of HIV-1 nucleic acids is indicative of active HIV-1 infection in infants born to HIV-infected mothers who have maternal antibodies to HIV-1. 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-A tests. 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.This test is intended for use in conjunction with clinical presentation and other laboratory markers of disease progression 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. en The cobas® HIV-1 quantitative test 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 sample preparation instrument and the cobas® z 480 real-time PCR 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 HIV 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 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 reagent 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 sample is achieved by the use of target virus-specific forward and reverse primers, which are selected from highly conserved regions of HIV. 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 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 deoxythymidine triphosphate (dTTP), which is incorporated into the newly synthesized DNA (amplicon).14-16 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, any newly formed amplicon is not eliminated since the AmpErase enzyme is inactivated once exposed to temperatures above 55°C. cobas® HIV-1 master mix contains two detection probes specific for the HIV-1 target sequences and one for 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 detection channels.17,18 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' 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. 14. Longo MC, Berninger MS, Hartley JL. Use of uracil DNA glycosylase to control carry-over contamination in polymerase chain reactions. Gene. 1990;93:125-128. 15. 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. 16. 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. 17. Higuchi R, Dollinger G, Walsh PS, Griffith R. Simultaneous amplification and detection of specific DNA sequences. Biotechnology (NY). 1992;10:413-417. 18. Heid CA, Stevens J, Livak JK, Williams PM. Real time quantitative PCR. Genome Res. 1996;6:986-994. en The cobas® HIV-1 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 sample preparation instrument and the cobas® z 480 real-time PCR 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 HIV 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 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 reagent 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 sample is achieved by the use of target virus-specific forward and reverse primers, which are selected from highly conserved regions of HIV. 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 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 deoxythymidine triphosphate (dTTP), which is incorporated into the newly synthesized DNA (amplicon).12-14 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, any newly formed amplicon is not eliminated since the AmpErase enzyme is inactivated once exposed to temperatures above 55°C. cobas® HIV-1 master mix contains two detection probes specific for the HIV-1 target sequences and one for 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 detection channels.15,16 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' 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. 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. 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. 14. 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. 15. Higuchi R, Dollinger G, Walsh PS, Griffith R. Simultaneous amplification and detection of specific DNA sequences. Biotechnology (NY). 1992;10:413-417. 16. Heid CA, Stevens J, Livak JK, Williams PM. Real time quantitative PCR. Genome Res. 1996;6:986-994. en

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