For in vitro diagnostic use. Others cobas Malaria 5800-6800-8800 IVD cobas® Malaria PID00000525 for use on the cobas® 5800/6800/8800 systems 09 352 511 190 9 352 511 190 09352511190 9352511190 09352511190 KIT COBAS 58/68/8800 MALARIA 192T KIT COBAS 58/68/8800 MALARIA 192T 00875197007183 Reagents, kits 1 kit 192 tests true The cobas® Malaria test is based on a fully automated sample preparation (nucleic acid extraction and purification) followed by PCR amplification and detection. The cobas® 5800 system consists of a single, 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 the cobas® 6800/8800 systems software, which assigns test results for all tests as either non- reactive, reactive or invalid. When using the cobas® 5800/6800/8800 systems, results can be reviewed directly on the system screen, printed as a report, or sent to a Laboratory Information Management System (LIMS) or other result management system. Samples can either be tested individually or, tested in pools consisting of multiple samples. If pooling is to be performed, the cobas® Synergy software with the Hamilton MICROLAB® STAR/STARlet IVD may be used. Whole blood may be collected in the designated Roche Whole Blood Collection Tube. Alternatively whole blood may be collected in EDTA anticoagulant and transferred manually to the Roche Whole Blood Collection Tube. The Roche Whole Blood Collection Tube contains a pre-analytic, guanidine-based, chaotropic reagent, used to lyse cells within the whole blood, releasing and preserving nucleic acids. The tube containing the lysed whole blood is the primary tube on the analyzer, on which the universal sample preparation steps will be performed by the cobas® 5800/6800/8800 systems. Armored RNA internal control (IC) molecules are added during universal sample preparation and serves as a full process control from sample preparation through amplification/detection. The IC monitors for interference that could cause false negative results. Potentially affected samples are invalidated. The test also utilizes two external controls: a positive and a negative control. In addition to the sample lysis and release of nucleic acid which occurs in the primary tube, nucleic acids are also released by addition of proteinase and lysis reagent to the sample and controls. The released nucleic acids bind to the silica surface of the magnetic glass particles, which are added to the sample. Unbound substances and impurities, such as denatured proteins, cellular debris, and potential PCR inhibitors (such as hemoglobin) are removed with subsequent wash reagent steps and purified nucleic acids are eluted from the glass particles with elution buffer at elevated temperature. Selective amplification of target nucleic acid from the sample is achieved by the use of specific forward and reverse primers which are selected from highly conserved regions of the target nucleic acid. A thermostable DNA polymerase enzyme is used for both reverse-transcription and amplification. The master mix includes deoxyuridine triphosphate (dUTP), instead of deoxythimidine triphosphate (dTTP), which is incorporated into the newly synthesized DNA (amplicon).10-12 Any contaminating amplicons from previous PCR runs are destroyed by the AmpErase enzyme [uracil-N- glycosylase], which is included in the PCR mix, when heated in the first thermal cycling step. Newly formed amplicons are not destroyed since the AmpErase enzyme is inactivated once exposed to temperatures above 55°C. The cobas® Malaria master mix contains detection probes which are specific for Plasmodium and IC nucleic acid. The specific Plasmodium and IC 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 reporter dyes is measured at a defined wavelength, thus permitting detection and discrimination of the amplified Plasmodium targets and the IC.13,14 When not bound to the target sequence, the fluorescent signal of the intact probes 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 by the 5' to 3' nuclease 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 dyes are concomitantly increased. Since the two specific reporter dyes are measured at defined wavelengths, simultaneous detection and discrimination of the amplified Plasmodium targets and the IC are possible. 10.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. 11. 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. 12. 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. 13. Higuchi R, Dollinger G, Walsh PS, Griffith R. Simultaneous amplification and detection of specific DNA sequences. Biotechnology (N Y). 1992;10:413-7. 14. Heid CA, Stevens J, Livak KJ, Williams PM. Real time quantitative PCR. Genome Res. 1996;6:986-94. en cobas® Malaria is based on a 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 Systems software, which assigns test results for all tests as either non-reactive, reactive or invalid. Results can be reviewed directly on the system screen, and printed as a report. Samples should be tested as individual samples. Whole blood may be collected in the designated Roche Whole Blood Collection Tube. Alternatively, whole blood may be collected in EDTA anticoagulant and transferred manually to the Roche Whole Blood Collection Tube. The Roche Whole Blood Collection Tube contains a pre-analytic, guanidine-based, chaotropic reagent, used to lyse cells within the whole blood, releasing and preserving nucleic acids. The tube containing the lysed whole blood is the primary tube on the analyzer, on which the universal sample preparation steps will be performed by the cobas® 6800/8800 Systems. Armored RNA internal control (IC) molecules are added during universal sample preparation and serves as a full process control from sample preparation through amplification/detection. The IC monitors for interference that could cause false negative results. Potentially affected samples are invalidated. The test also utilizes two external controls: a positive and a negative control. In addition to the sample lysis and release of nucleic acid which occurs in the primary tube, nucleic acids are also released by addition of proteinase and lysis reagent to the sample and controls. The released nucleic acids bind to the silica surface of the magnetic glass particles, which are added to the sample. Unbound substances and impurities, such as denatured proteins, cellular debris, and potential PCR inhibitors (such as hemoglobin) are removed with subsequent wash reagent steps and purified nucleic acids are eluted from the glass particles with elution buffer at elevated temperature. Selective amplification of target nucleic acid from the donor sample is achieved by the use of specific forward and reverse primers which are selected from highly conserved regions of the target nucleic acid. A thermostable DNA polymerase enzyme is used for both reverse-transcription and amplification. The master mix includes deoxyuridine triphosphate (dUTP), instead of deoxythimidine triphosphate (dTTP), which is incorporated into the newly synthesized DNA (amplicon).10-12 Any contaminating amplicons from previous PCR runs are destroyed by the AmpErase enzyme [uracil-N- glycosylase], which is included in the PCR mix, when heated in the first thermal cycling step. Newly formed amplicons are not destroyed since the AmpErase enzyme is inactivated once exposed to temperatures above 55°C. The cobas® Malaria master mix contains detection probes which are specific for Plasmodium and IC nucleic acid. The specific Plasmodium and IC 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 reporter dyes is measured at a defined wavelength, thus permitting detection and discrimination of the amplified Plasmodium targets and the IC.13,14 When not bound to the target sequence, the fluorescent signal of the intact probes 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 by the 5' to 3' nuclease 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 dyes are concomitantly increased. Since the two specific reporter dyes are measured at defined wavelengths, simultaneous detection and discrimination of the amplified Plasmodium targets and the IC are possible. 10. 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. 11. 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. 12. 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. 13. Higuchi R, Dollinger G, Walsh PS, Griffith R. Simultaneous amplification and detection of specific DNA sequences. Biotechnology (N Y). 1992;10:413-7. 14. Heid CA, Stevens J, Livak KJ, Williams PM. Real time quantitative PCR. Genome Res. 1996;6:986-94. en The cobas® Malaria test for use on the cobas® 5800/6800/8800 Systems (cobas® Malaria) is a qualitative in vitro nucleic acid screening test for the direct detection of Plasmodium (P. falciparum, P. malariae, P. vivax, P. ovale and P. knowlesi) DNA and RNA in whole blood samples from individual human donors, including donors of whole blood and blood components, as well as other living donors. It is also intended for use in testing whole blood samples to screen organ and tissue donors when samples are obtained while the donor’s heart is still beating. Whole blood samples from all donors may be screened as individual samples. For donations of whole blood and blood components, whole blood samples may be tested individually or in pools comprised of aliquots of individual samples. The test is not intended for use as an aid in diagnosis of Plasmodium infection. This test is not intended for use on samples of cord blood. This test is not intended for use on cadaveric samples. en The cobas® Malaria test for use on the cobas® 5800/6800/8800 systems (cobas® Malaria) is a qualitative in vitro nucleic acid screening test for the direct detection of Plasmodium (P. falciparum, P. malariae, P. vivax, P. ovale and P. knowlesi) DNA and RNA in whole blood samples from individual human donors, including donors of whole blood and blood components, as well as other living donors. It is also intended for use in testing whole blood samples to screen organ and tissue donors when samples are obtained while the donor’s heart is still beating. Whole blood samples from all donors may be screened as individual samples. For donations of whole blood and blood components, whole blood samples may be tested individually or in pools comprised of aliquots of individual samples. This test is not intended for use on samples of cord blood. This test is not intended for use on cadaveric samples. This test may also be used as an aid in diagnosis for Plasmodium infection in samples collected from individuals suspected of infection with Plasmodium parasite by their healthcare provider. en

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