For in vitro diagnostic use. Others cobas CHIKV DENV 5800 6800 8800 IVD cobas® CHIKV/DENV PID00000368 Nucleic acid test for use on the cobas® 5800/6800/8800 systems 09 040 650 190 9 040 650 190 09040650190 9040650190 09040650190 KIT C58/68/88 CHIKV/DENV 480T CE-IVD KIT C58/68/88 CHIKV/DENV 480T CE-IVD 00875197006544 Reagents, kits 1 Kit 480 tests true The cobas® CHIKV/DENV for use on the cobas® 5800/6800/8800 systems is a qualitative in vitro test for the direct detection of chikungunya virus (CHIKV) RNA and dengue virus (DENV) serotypes 1-4 RNA in human plasma. The test is intended for use to screen donor samples for CHIKV RNA or DENV RNA alone or to simultaneously screen for both CHIKV and DENV RNA in plasma from individual human donors, including donors of whole blood, blood components, and other living donors. This test is also intended for use to screen organ and tissue donors when donor samples are obtained while the donor’s heart is still beating. 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 of samples of cord blood. This test may also be used as an aid in diagnosis for CHIKV or DENV in samples collected from individuals suspected of infection with chikungunya or dengue viruses by their healthcare provider. When used as an aid in diagnosis, plasma samples should only be tested indivi en The cobas® CHIKV/DENV test is based 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 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. For donor screening, samples can either be tested individually or tested in pools consisting of multiple samples. If pooling is to be performed, the cobas® p 680 instrument (for cobas® 6800/8800 systems), or cobas® Synergy software with the Hamilton MICROLAB® STAR/STARlet IVD, may opt ionally be used in a pre-analytical step if pooling is to be performed. Nucleic acids from the sample and added armored RNA internal control (IC) molecules (which serve as a full process control from sample preparation through amplification/detection process control) are simultaneously extracted. The IC monitors for interference that could cause false negative results. Potentially affected samples are invalidated. In addition, the test utilizes two external controls: a positive and a negative control. 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 a dded magnetic glass particles. 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 g lass 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 nu cleic 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). 27-29 Any contamina ting amplicons from previous PCR runs are eliminated by the AmpErase enzyme [uracil- N-glycosylase], which is included in the PCR mix, when heated in the first thermal cycling step. However, newly formed amplicons are not destroyed since the AmpErase enzyme is inactivated once exposed to temperatures above 55°C. The cobas® CHIKV/DENV master mix contains detection probes which are specific for CHIKV, DENV and IC nucleic acid. The specific CHIKV, DENV and IC detection probes are each labeled with one of three unique fluorescent dyes which act as a reporter. Each probe also has a fourth dye which acts as a quencher. The three reporter dyes are measured at defined wavelengths, thus permitting simultaneous detection and discrimination of the amplified CHIKV and DENV targets and the IC. 30, 31 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 dye is concomitantly increased. Since the three specific reporter dyes are measured at defined wavelengths, simultaneous detection and discrimination of the amplified CHIKV and DENV targets and the IC are possible. 27. 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. 28. 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. 29. 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. 30. Higuchi R, Dollinger G, Walsh PS, Griffith R. Simultaneous amplification and detection of specific DNA sequences. Biotechnology (NY) 1992;10:413-417. 31. Heid CA, Stevens J, Livak JK, Williams PM. Real-time quantitative PCR. Genome Res 1996;986-994. en

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