For in vitro diagnostic use. Others cobas CT NG 58-68-88 IVD cobas® CT/NG PID00000154 Qualitative nucleic acid test for use on the cobas® 5800/6800/8800 systems 09 040 501 190 9 040 501 190 09040501190 9040501190 09040501190 KIT COBAS 58/68/8800 CT/NG 480T IVD cobas CT/NG 00875197006865 Reagents, kits 1 kit 480 tests true CE-IVD and Canada-IVD cobas® CT/NG for use on the cobas® 5800/6800/8800 systems is an automated, qualitative in vitro diagnostic test, that utilizes real-time polymerase chain reaction (PCR), for the direct detection of Chlamydia trachomatis (CT) and/or Neisseria gonorrhoeae (NG) DNA in male and female urine, clinician-instructed self-collected vaginal swab specimens, clinician-collected vaginal swab specimens, endocervical swab specimens, oropharyngeal (throat) swab specimens, and anorectal swab specimens, all collected in cobas® PCR Media (Roche Molecular Systems, Inc.), and cervical specimens collected in PreservCyt® Solution. This test is intended as an aid in the diagnosis of chlamydial and gonococcal disease in both symptomatic and asymptomatic individuals. en cobas® CT/NG for use on the cobas® 5800/6800/8800 Systems is an automated, qualitative in vitro nucleic acid diagnostic test, that utilizes real-time polymerase chain reaction (PCR), for the direct detection of Chlamydia trachomatis (CT) and/or Neisseria gonorrhoeae (NG) DNA in male and female urine, clinician-instructed self-collected vaginal swab specimens (collected in a clinical setting), and clinician-collected vaginal swab specimens, endocervical swab specimens, oropharyngeal (throat) swab specimens and anorectal swab specimens all collected in cobas® PCR Media (Roche Molecular Systems, Inc.), and cervical specimens collected in PreservCyt® Solution. This test is intended as an aid in the diagnosis of chlamydial and gonococcal disease in both symptomatic and asymptomatic individuals. en CE-IVD and Canada-IVD cobas® CT/NG 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 system or cobas® 6800/8800 systems software which assigns test results for all tests as positive, negative or invalid. Results can be reviewed directly on the system screen, exported, or printed as a report. Nucleic acid from patient samples, external controls and added internal control DNA (DNA-IC) molecules is simultaneously extracted. In summary, bacterial 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 steps and purified nucleic acid is 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-specific forward and reverse primers which are selected from highly conserved plasmid and genomic regions of CT and NG. A region on the CT cryptic plasmid and the ompA gene (dual target) and two conserved sequences of the NG DR-9 region are amplified by cobas® CT/NG. Selective amplification of DNA IC is achieved by the use of sequence-specific forward and reverse primers which are selected to have no homology with either the CT or NG target regions. A thermostable DNA polymerase enzyme is used for PCR amplification. The target and DNA-IC 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 are eliminated by the AmpErase enzyme, which is included in the PCR master mix, during the first thermal cycling step.16 However, newly formed amplicon are not eliminated since the AmpErase enzyme is inactivated once exposed to temperatures above 55°C. The cobas® CT/NG master mix contains two detection probes specific for the CT target sequences, two detection probes specific for the NG target sequences and one for the DNA-IC. The probes are labeled with target specific fluorescent reporter dyes allowing simultaneous detection of CT targets, NG targets and DNA-IC in three different target channels.17,18 When not bound to the target sequence, the fluorescent signal of the intact probes is 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' exonuclease 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 CT and NG targets and DNA-IC, respectively. 16. 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. 17. Higuchi R, Dollinger G, Walsh PS, Griffith R. Simultaneous amplification and detection of specific DNA sequences. Biotechnology (NY). 1992; 10:413-7. 18. Heid CA, Stevens J, Livak JK, Williams PM. Real time quantitative PCR. Genome Res. 1996; 6:986-94. en cobas® CT/NG 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 System or cobas® 6800/8800 Systems software which assigns test results for all tests as positive, negative or invalid. Results can be reviewed directly on the system screen, exported, or printed as a report.Nucleic acid from patient samples and added internal control DNA (DNA-IC) molecules is simultaneously extracted. In summary, bacterial 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 steps and purified nucleic acid is eluted from the magnetic glass particles with elution buffer at elevated temperature. External controls (positive and negative) are processed in the same way with each cobas® CT/NG run.Selective amplification of target nucleic acid from the sample is achieved by the use of target-specific forward and reverse primers which are selected from highly conserved plasmid and genomic regions of CT and NG. A region on the CT cryptic plasmid and the ompA gene (dual target) and two conserved sequences of the NG DR-9 region are amplified by cobas® CT/NG. Selective amplification of DNA-IC is achieved by the use of sequence-specific forward and reverse primers which are selected to have no homology with either the CT or NG target regions. A thermostable DNA polymerase enzyme is used for PCR amplification. The target and DNA-IC 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 amplicons from previous PCR runs are eliminated by the AmpErase enzyme, which is included in the PCR master mix, during the first thermal cycling step.16 However, newly formed amplicons are not eliminated since the AmpErase enzyme is inactivated once exposed to temperatures above 55°C.The cobas® CT/NG master mix contains two detection probes specific for the CT target sequences, two detection probes specific for the NG target sequences and one for the DNA-IC. The probes are labeled with target specific fluorescent reporter dyes allowing simultaneous detection of CT targets, NG targets and DNA-IC in three different target channels.17,18 When not bound to the target sequence, the fluorescent signal of the intact probes is 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' exonuclease 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 CT and NG targets and DNA-IC, respectively.16. Longo MC, Berninger MS, Hartley JL. Use of uracil DNA glycosylase to control carry-over contamination in polymerase chain reactions. Gene. 1990; 93:125-8.17. Higuchi R, Dollinger G, Walsh PS, Griffith R. Simultaneous amplification and detection of specific DNA sequences. Biotechnology (NY). 1992; 10:413-7.18. Heid CA, Stevens J, Livak JK, Williams PM. Real time quantitative PCR. Genome Res. 1996; 6:986-94. en

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