For in vitro diagnostic use. Others cobas MTB-RIF INH 58 68 88 IVD cobas® MTB-RIF/INH PID00000332 for use on the cobas® 5800/6800/8800 Systems 09040617190 KIT COBAS 58/68/8800 MTB-RIF/INH 192T KIT COBAS 58/68/8800 MTB-RIF/INH 192T 00875197006643 Reagents, kits 1 Kit 192 tests true cobas® MTB-RIF/INH 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 rifampicin-resistance associated mutations of the rpoB gene, and isoniazid-resistance associated mutations in the katG and inhA genes, of Mycobacterium tuberculosis, from human respiratory specimens. The test is intended for use on raw sputum, and digested and decontaminated (N-acetyl-L-cysteine/ NaOH treated) sputum and bronchial alveolar lavage (BAL) samples, that tested positive for Mycobacterium tuberculosis complex (MTBC) by cobas® MTB. This test is intended for use in conjunction with culture and drug susceptibility testing, and as a reflex test together with cobas® MTB, as an aid in the diagnosis of infection with a multidrug resistant M. tuberculosis (MDR-TB). en cobas® MTB-RIF/INH is based on pre-analytic sample liquefaction and mycobacterial inactivation followed by sample sonication and fully automated sample preparation (nucleic acid extraction and purification) and PCR amplification and detection. Sample liquefaction and mycobacterial 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 System 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 external controls is simultaneously extracted. In summary, bacterial nucleic acid is released by chemical (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 and detection of target nucleic acid from the sample is achieved by the use of target gene specific forward and reverse primers. Eighteen Rifampicin-resistance associated mutations (rpoB gene) are detected by one set of primer and multiple mutation-specific probes. Seven Isoniazid-resistance associated mutations (katG gene and inhA gene promoter region) are detected by two sets of primers and multiple mutation-specific probes. MTB is detected by one set of primers and one probe targeting a highly conserved single-copy genomic sequence of the MTB complex. A thermostable DNA polymerase enzyme is used for PCR amplification. The target 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 amplicon are not eliminated since the AmpErase enzyme is inactivated once exposed to temperatures above 55°C. The cobas® MTB-RIF/INH master mix contains multiple probes specific for Rifampicin-resistance associated mutations and Isoniazid-resistance associated mutations and one probe specific for the MTB complex. The probes are labeled with target specific fluorescent reporter dyes allowing simultaneous detection of Rifampicin-resistance associated mutations, Isoniazid-resistance associated mutations and the MTB complex target in five different target channels (Rifampicin-resistance associated mutations are detected in three of the five detection 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 Rifampicin-resistance associated mutations, Isoniazid-resistance associated mutations and MTB complex target, 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