For in vitro diagnostic use. Others cobas MTB 58 68 88 IVD cobas® MTB PID00000333 for use on the cobas® 5800/6800/8800 Systems 09 040 579 190 9 040 579 190 09040579190 9040579190 09040579190 KIT COBAS 58/68/8800 MTB 384T KIT COBAS 58/68/8800 MTB 384T 00875197006605 Reagents, kits 1 Kit 384 tests true cobas® MTB 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 Mycobacterium tuberculosis complex (MTBC) DNA, in human respiratory specimens; including raw sputum, and digested and decontaminated (N-acetyl-L- cysteine/NaOH [NALC-NaOH]-treated) sputum and bronchoalveolar lavage (BAL) samples. This test is for use with specimens from patients who are suspected of Mycobacterium tuberculosis infection, and who are not taking antituberculosis therapy. This test is intended as an aid in the diagnosis of pulmonary tuberculosis, and in conjunction with other laboratory findings, as well as clinical signs and symptoms. en cobas® MTB is based on pre-analytic sample liquefaction and mycobacteria inactivation followed by sample sonication and fully automated sample preparation (nucleic acid extraction and purification) and PCR amplification and detection. Sample liquefaction and mycobacteria inactivation occur simultaneously during sample incubation with cobas® Microbial Inactivation Solution (MIS). Sonication of liquefied and inactivated sample is performed prior to loading onto the cobas® 5800/6800/8800 Systems. 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 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 chemical (cobas® Microbial Inactivation Solution [MIS], cobas® omni Lysis Reagent), enzymatic (proteinase), and physical (sonication) disruption of bacteria. 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 for the MTB complex which are selected from highly-conserved regions within the respective target organism. MTB is detected by two selective sets of primers and two probes targeting separate regions (dual-target, 16S rRNA gene and esx genes - esxJ, esxK, esxM, esxP, and esxW). 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 the MTB complex 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 deoxythymidine 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.9 However, newly formed amplicons are not eliminated since the AmpErase enzyme is inactivated once exposed to temperatures above 55°C. The cobas® MTB master mix contains two detection probes specific for the MTB complex target sequences and one for the DNA-IC. The target specific probes are labeled with different fluorescent reporter dyes allowing simultaneous detection of MTB complex target and DNA-IC in two different target channels.10,11 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 causing the 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 MTB complex targets and DNA-IC, respectively. 9. 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. 10. Higuchi R, Dollinger G, Walsh PS, Griffith R. Simultaneous amplification and detection of specific DNA sequences. Biotechnology (N Y). 1992;10:413-7. 11. Heid CA, Stevens J, Livak KJ, Williams PM. Real time quantitative PCR. Genome Res. 1996;6:986-94. en

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