For in vitro diagnostic use. Others cobas CMV Test 5800-6800-8800 IVD cobas® CMV RMD-58-68-88-CMV Quantitative nucleic acid test for use on the cobas® 5800/6800/8800 systems 09 040 897 190 9 040 897 190 09040897190 9040897190 09040897190 KIT COBAS 58/68/8800 CMV 192T IVD cobas CMV 00875197006421 Reagents, kits 1 kit 192 tests true cobas® CMV is an in vitro nucleic acid amplification test for the quantitation 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 an in vitro nucleic acid amplification test for the quantitation of cytomegalovirus (CMV) DNA in human EDTA plasma. cobas® CMV is intended for use as an aid in the management of CMV in solid organ transplant patients and in hematopoietic stem cell transplant patients. 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.cobas® CMV is not intended for use as a screening test for blood or blood products. en cobas® CMV is an in vitro nucleic acid amplification test for the quantitation of cytomegalovirus (CMV) DNA in human EDTA plasma.cobas® CMV is intended for use as an aid in the 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 US-IVD and CE-IVDcobas® CMV is based on fully automated sample preparation (nucleic acid extraction and purification) followed by PCR amplification and detection. The cobas® 5800 System is designed as one 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 either target not detected, CMV DNA detected < LLoQ (lower limit of quantitation), CMV DNA detected > ULoQ (upper limit of quantitation), or 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 acid from patient samples and added lambda DNA-QS molecules is simultaneously extracted. In summary, 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 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 target virus-specific forward and reverse primers which are selected from highly-conserved regions of the CMV DNA polymerase (UL54) gene. Selective amplificationof 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). 21,22,23 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. However, newly formed amplicon are not eliminated since the AmpErase enzyme is inactivated once exposed to temperatures above 55°C.The cobas® CMV master mix contains one detection probe specific for 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 target channels.24,25 The fluorescent signal of the intact probes is suppressed by the quencher dye. During the PCR amplification step, hybridization of the probe to the specific single-stranded DNA templates 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. 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, Brown T, Pearl L. The structural basis of specific base-excision repair by uracil-DNA glycosylase. Nature. 1995;373:487-93.23. 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;80:869-78.24. Higuchi R, Dollinger G, Walsh PS, Griffith R. Simultaneous amplification and detection of specific DNA sequences. Biotechnology (N Y). 1992;10:413-7.25. Heid CA, Stevens J, Livak KJ, Williams PM. Real time quantitative PCR. Genome Res. 1996;6:986-94. en cobas®CMV 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 either target not detected, CMV DNA detected < LLoQ (lower limit of quantitation), CMV DNA detected > ULoQ (upper limit of quantitation), or 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 acid from patient samples and added lambda DNA-QS molecules is simultaneously extracted. In summary, 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 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 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).23,24,25 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. However, newly formed amplicon are not eliminated since the AmpErase enzyme is inactivated once exposed to temperatures above 55°C.The cobas® CMV master mix contains one detection probe specific for 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 target channels.26,27 The fluorescent signal of the intact probes is suppressed by the quencher dye. During the PCR amplification step, hybridization of the probe to the specific single-stranded DNA templates 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. 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.23. Longo MC, Berninger MS, Hartley JL. Use of uracil DNA glycosylase to control carry-over contamination in polymerase chain reactions. Gene. 1990;93:125-128.24. Savva R, McAuley-Hecht K, Brown T, Pearl L. The structural basis of specific base-excision repair by uracil-DNA glycosylase. Nature. 1995;373:487-493.25. Mol CD, Arvai AS, Slupphau G, et al. Crystal structure and mutational analysis of human uracil-DNA glycosylase: structural basis for specificity and catalysis. Cell. 1995;80:869-878.26. Higuchi R, Dollinger G, Walsh PS, Griffith R. Simultaneous amplification and detection of specific DNA sequences. Biotechnology (NY). 1992;10:413-417.27. Heid CA, Stevens J, Livak JK, Williams PM. Real time quantitative PCR. Genome Res. 1996;6:986-994. en

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