For in vitro diagnostic use. Others cobas HEV 5800-6800-8800 IVD cobas® HEV PID00000526 Nucleic acid test for use on the cobas® 5800/6800/8800 systems 09 040 986 190 9 040 986 190 09040986190 9040986190 09040986190 KIT COBAS 58/68/8800 HEV 192T CE-IVD KIT COBAS 58/68/8800 HEV 192T CE-IVD 00875197006704 Reagents, kits 1 kit 192 tests true The cobas® HEV test is based on real time PCR technology on a fully automated sample preparation (nucleic acid extraction and purification) followed by PCR amplification and detection system. 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 cobas® 6800/8800 software which assigns test results for all tests as non-reactive, reactive, or invalid. When using the cobas® 5800 System, the cobas® Synergy software is recommended for reviewing results and printing reports and it is required to send results to a LIMS or other result management system. When using the cobas® 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.When testing on cobas® 5800 Systems, use of the cobas® Synergy software is required even if pooling is not performed. If pooling in a pre-analytical step is performed, cobas® Synergy software with the Hamilton MICROLAB® STAR/STARlet IVD may be used.When testing on the cobas® 6800/8800 Systems, the cobas p 680 instrument, or cobas® Synergy software with the Hamilton MICROLAB® STAR/STARlet IVD, may optionally be used in a pre-analytical step if pooling is to be performed.Nucleic acid from the sample and added armored RNA internal control (IC) molecules (which serve as the sample preparation and amplification/detection process control) is simultaneously extracted. In addition the test utilizes two kit controls: a positive and a negative control. 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 (such as hemoglobin) are removed with subsequent wash reagent steps and purified nucleic acid is 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 virus-specific forward and reverse primers which are selected from highly conserved regions of the viral 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). 50-52 Any contaminating amplicon from previous PCR run is destroyed by the AmpErase enzyme [uracil-N-glycosylase], which is included in the PCR master mix, when heated in the first thermal cycling step. However, newly formed amplicon is not destroyed since the AmpErase enzyme is inactivated once exposed to temperatures above 55°C.The cobas® HEV master mix contains detection probes which are specific for HEV and IC nucleic acid. The specific HEV 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 two reporter dyes are measured at defined wavelengths, thus permitting simultaneous detection and discrimination of the amplified HEV target and the IC. 53,54 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 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 dye is concomitantly increased. Since the two specific reporter dyes are measured at defined wavelengths, simultaneous detection and discrimination of the amplified HEV target and the IC are possible.50. Longo MC, Berninger MS, Hartley JL. Use of uracil DNA glycosylase to control carry-over contamination in polymerase chain reactions. Gene. 1990;93:125-128.51. Savva R, McAuley-Hecht K, Brown T, Pearl L. The structural basis of specific base-excision repair by uracil-DNA glycosylase. Nature. 1995;373:487-493.52. 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;80:869-878.53. Higuchi R, Dollinger G, Walsh PS, Griffith R. Simultaneous amplification and detection of specific DNA sequences. Biotechnology (NY). 1992;10:413-417.54. Heid CA, Stevens J, Livak JK, Williams PM. Real-time quantitative PCR. Genome Res 1996;986-994. en The cobas® HEV test is based on real time PCR technology on a fully automated sample preparation (nucleic acid extraction and purification) followed by PCR amplification and detection on the system. 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 6800/8800 system software which assigns test results for all tests as non-reactive, reactive, or invalid. When using the cobas® 5800/6800/8800 systems, results can be reviewed directly on the system screen, and 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, cobas® Synergy software with the Hamilton MICROLAB® STAR/STARlet IVD may be used. Nucleic acid from the sample and added armored RNA internal control (IC) molecules (which serve as a full process control from sample preparation and through amplification/detection) is simultaneously extracted. The IC monitors for interference that could cause false negative results. Potentially affected samples are invalidated. In addition the test utilizes two kit controls: a positive and a negative control. 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 (such as hemoglobin) are removed with subsequent wash reagent steps and purified nucleic acid is 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 virus-specific forward and reverse primers which are selected from highly conserved regions of the viral 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).49-51 Any contaminating amplicon from previous PCR run is destroyed by the AmpErase enzyme [uracil-N-glycosylase], which is included in the PCR master mix, when heated in the first thermal cycling step. However, newly formed amplicon is not destroyed since the AmpErase enzyme is inactivated once exposed to temperatures above 55°C. The cobas® HEV master mix contains detection probes which are specific for HEV and IC nucleic acid. The specific HEV 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 two reporter dyes are measured at defined wavelengths, thus permitting simultaneous detection and discrimination of the amplified HEV target and the IC.52,53 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 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 dye is concomitantly increased. Since the two specific reporter dyes are measured at defined wavelengths, simultaneous detection and discrimination of the amplified HEV target and the IC are possible 49. 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. 50. 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. 51. 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. 52. Higuchi R, Dollinger G, Walsh PS, Griffith R. Simultaneous amplification and detection of specific DNA sequences. Biotechnology (N Y). 1992;10:413-7. 53. Heid CA, Stevens J, Livak KJ, Williams PM. Real time quantitative PCR. Genome Res. 1996;6:986-94. en The cobas® HEV test for use on cobas® 5800/6800/8800 Systems is a qualitative in vitro nucleic acid amplification test for the direct detection of hepatitis E virus (HEV) RNA (genotypes 1-4) in human plasma. This test is intended for use to screen donor samples for HEV RNA in plasma samples from individual human donors, including donors of whole blood, blood components (red cells, platelets, and plasma), and other living donors. Plasma from all donors may be screened as individual samples. For donations of whole blood and blood components, plasma samples may be tested individually or plasma may be tested 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 as an aid in diagnosis for HEV. en The cobas® HEV test for use on cobas® 5800/6800/8800 systems is a qualitative in vitro nucleic acid amplification test for the direct detection of hepatitis E virus (HEV) RNA (genotypes 1-4) in human plasma. This test is intended for use to screen donor samples for HEV RNA in plasma samples from individual human donors, including donors of whole blood, blood components (red cells, platelets, and plasma), and other living donors. Plasma from all donors may be screened as individual samples. For donations of whole blood and blood components, plasma samples may be tested individually or plasma may be tested in pools comprised of aliquots of individual samples. This test is not intended for use on samples of cord blood. This test may also be used as an aid in diagnosis for HEV in samples collected from individuals suspected of infection with HEV virus by their healthcare provider. en

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