For in vitro diagnostic use. Others cobas CMV Test 4800 IVD cobas® CMV Test RMD-4800-CMV-001 Quantitative nucleic acid test for use on the cobas® 4800 system 07 865 970 190 7 865 970 190 07865970190 7865970190 07865970190 KIT COBAS 4800 CMV 120T CE-IVD cobas CMV Test 00875197005882 Reagents, kits 1 kit 120 tests true cobas® CMV is an in-vitro nucleic acid amplification test for the quantitative measurement of cytomegalovirus (CMV) DNA in human EDTA plasma. cobas® CMV is intended for use as an aid in the diagnosis and management of CMV in solid organ transplant patients and in hematopoietic stem cell transplant patients. The test can be used in these populations to assess the need to initiate antiviral treatment. In patients receiving anti-CMV therapy, serial DNA measurements can be used to assess viral response to treatment. The results from cobas® CMV must be interpreted within the context of all relevant clinical and laboratory findings. en cobas® CMV 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 instrument and the cobas® z 480 analyzer. Automated data management is performed by the cobas® 4800 software which assigns test results for all tests as target not detected, < LLoQ (below lower limit of quantitation), > ULoQ (above upper limit of quantitation) or CMV DNA 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 added lambda DNA 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 buffer steps and purified nucleic acids are eluted from the magnetic glass particles with elution buffer at elevated temperature. Selective amplification of target nucleic acid from the sample is achieved by the use of target virus-specific forward and reverse primers which are selected from highly-conserved regions of the CMV DNA polymerase (UL54) gene. Selective amplification of DNA QS is achieved by the use of sequence-specific forward and reverse primers which are selected to have no homology with the CMV genome. A thermostable DNA polymerase enzyme is used for amplification. The target and DNA 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 deoxythimidine triphosphate (dTTP), which is incorporated into the newly synthesized DNA (amplicon). Any contaminating amplicon from previous PCR runs is eliminated by the AmpErase enzyme, which is included in the PCR mix, when heated in the first thermal cycling step.21-23 However, newly formed amplicon are not eliminated since the AmpErase enzyme is inactivated once exposed to temperatures above 55°C. cobas® CMV master mix contains one detection probe specific for the CMV target sequences and one for the DNA QS. The probes are labeled with target-specific fluorescent reporter dyes allowing simultaneous detection of CMV target and DNA QS in two different detection channels.24, 25 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' 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 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 DNA QS. 21. 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. 22. Savva R, McAuley-Hecht K, BrownT, PearlL. The structural basis of specific base-excisionrepair byuracil-DNA glycosylase. Nature. 1995;373:487-93. 23. Mol CD, Arvai AS, Slupphau G, et al.Crystalstructure and mutationalanalysis of humanuracil-DNA glycosylase: structural basis for specificity and catalysis. Cell. 1995;80:869-78. 24. Higuchi R, Dollinger G, Walsh PS, Griffith R.Simultaneous amplification anddetection of specific DNA sequences. Biotechnology (NY).1992;10:413-7. 25. Heid CA, Stevens J, LivakKJ, Williams PM. Real time quantitative PCR. Genome Res. 1996;6:986-94. en

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