For in vitro diagnostic use. Others cobas CHIKV DENV IVD cobas® CHIKV/DENV RMD-6800-8800-CHIKV-001 Nucleic acid test for use on the cobas® 6800/8800 systems 08 042 276 190 8 042 276 190 08042276190 8042276190 08042276190 KIT C68/88 CHIKV/DENV 480T CE-IVD cobas CHIKV/DENV 00875197005929 Reagents, kits 1 kit 480 tests true cobas® CHIKV/DENV for use on the cobas® 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 is not intended for use as an aid in diagnosis for CHIKV or DENV. en cobas® CHIKV/DENV is based on 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 software which assigns test results for all tests as non-reactive, reactive, or invalid. Results can be reviewed directly on the system screen, and printed as a report.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, or cobas® Synergy Software with the Hamilton MICROLAB® STAR IVD (cobas® Synergy Core), may optionally 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 the sample preparation and amplification/detection process control) are simultaneously extracted. 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 added 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 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).27-29 Any contaminating 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 CHIKVand 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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