For in vitro diagnostic use. Others cobas 4800 CT NG v2.0 IVD cobas® 4800 CT/NG v2.0 RMD-4800-CTNG-006 07102569190 KIT C4800 CT/NG AMP DET 240T V2.0 US-IVD cobas 4800 CT/NG v2.0 00875197005356 Reagents, kits 1 kit 240 tests true 07102577190 KIT C4800 CT/NG AMP DET 960T V2.0 US-IVD cobas 4800 CT/NG v2.0 00875197005363 Reagents, kits 1 kit 960 tests true The cobas® CT/NG v2.0 Test is an automated, in vitro nucleic acid amplification test for the qualitative detection of Chlamydia trachomatis (CT) and/or Neisseria gonorrhoeae (NG) DNA in urogenital specimens. The Test utilizes the Polymerase Chain Reaction (PCR) for the detection of Chlamydia trachomatis and Neisseria gonorrhoeae DNA in male and female urine, self-collected vaginal swab specimens (collected in a clinical setting) clinician-collected vaginal swab specimens, and endocervical 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 The cobas® CT/NG v2.0 Test for Chlamydia trachomatis and Neisseria gonorrhoeae is based on 2 major processes: (1) automated sample preparation to obtain nucleic acids, including CT and NG DNA; (2) simultaneous PCR amplification of target DNA sequences using both CT and NG specific primer pairs and real-time detection of cleaved fluorescent-labeled CT and NG specific oligonucleotide detection probes. An Internal Control, containing CT and NG DNA, is added to all samples prior to automated sample preparation and is amplified and detected simultaneously with each sample to monitor the entire process. Sample preparation for the cobas® CT/NG v2.0 Test is automated with the use of the cobas® x 480 instrument, which is part of the cobas® 4800 System. Specimens are lysed in the collection device by the chaotropic agent in the cobas® PCR Media. Released nucleic acids, along with added CT/NG Internal Control DNA, are purified through binding to magnetic glass particles, washed, and finally separated from these particles making them ready for PCR amplification and detection. The Master Mix reagent contains primer pairs and probes specific for the CT cryptic plasmid DNA, the CT genomic ompA gene DNA, NG genomic DNA sequences A and B within the DR-9 region, and CT and NG Internal Control DNA. PCR Amplification Target Selection In addition to chromosomal DNA, C. trachomatis contains an approximately 7,500 base pair cryptic plasmid that is common to all serovars of C. trachomatis. 9,10 The cobas® CT/NG v2.0 Test uses the CT primers CP102 and CP103 to define a sequence of approximately 206 nucleotides within the cryptic plasmid DNA of C. trachomatis. In addition, the cobas® CT/NG v2.0 Test uses the CT primers CTMP101 and CTMP102 to define a sequence of approximately 182 nucleotides within the chromosomal DNA of C. trachomatis. The N. gonorrhoeae target site is a highly conserved direct repeat region called DR-9. The cobas® CT/NG v2.0 Test uses the NG primers NG514 and NG519 to define a sequence of approximately 190 nucleotides (DR-9A) from this region. In addition, the cobas® CT/NG v2.0 Test uses another set of NG primers, NG552 and NG579, to define a second sequence of approximately 215 nucleotides (DR-9B) from this region. Target Amplification Processed samples are added to the amplification mixture in a microwell plate by the cobas® x 480 instrument. The microwell plate is then transferred to the cobas® z 480 analyzer, where PCR amplification occurs. The reaction mixture is heated to separate the isolated double-stranded DNA and expose the primer target sequences. As the mixture cools, the primers anneal to the target DNA. Z05 DNA polymerase, in the presence of Mn2+ and excess dNTPs, extends the annealed primers along the target templates to produce double-stranded DNA. This completes the first cycle of PCR, yielding a double-stranded DNA copy of the target regions of the CT and/or NG DNA and the CT/NG Internal Control DNA. Repetition of this process results in the amplification of DNA between ´the primer target sequences, producing a double-stranded DNA molecule termed an amplicon. The cobas® z 480 analyzer automatically repeats this process for a designated number of cycles, with each cycle intended to double the amount of amplicon DNA. The required number of cycles is preprogrammed into the cobas® 4800 Software. Amplification occurs only in the specific CT and/or NG targets between their respective primers; the entire CT cryptic plasmid or CT and/or NG genomes are not amplified. Internal Control Amplification The CT/NG Internal Control is a combination of two non-infectious recombinant plasmid DNAs, each with primer binding regions identical to those of either the C. trachomatis or the N. gonorrhoeae genomic target sequences. Both recombinant plasmid DNAs have an identical randomized internal target sequence, and a unique probe binding region that differentiates the CT/NG Internal Control from target amplicon. These features were selected to ensure independent detection of both the CT/NG Internal Control and the C. trachomatis and N. gonorrhoeae target DNAs. The CT/NG Internal Control Reagent is included in the cobas® CT/NG v2.0 Test and is introduced into each sample on the cobas® x 480 instrument during sample processing. Selective Amplification Selective amplification of target nucleic acid from the specimen is achieved in the cobas® CT/NG v2.0 Test by the use of AmpErase® Uracil-N- Glycosylase (UNG) enzyme and deoxyuridine triphosphate (dUTP). The AmpErase enzyme recognizes and catalyzes the destruction of DNA strands containing deoxyuridine11, but not DNA containing deoxythymidine. Deoxyuridine is not present in naturally occurring DNA, but is always present in amplicon due to the use of deoxyuridine triphosphate in place of thymidine triphosphate as one of the dNTPs in the Master Mix reagent; therefore, only amplicon contain deoxyuridine. Deoxyuridine renders contaminating amplicon susceptible to destruction by AmpErase enzyme prior to amplification of the target DNA. AmpErase enzyme, which is included in the Master Mix reagent, catalyzes the cleavage of deoxyuridine-containing DNA at the deoxyuridine residues by opening the deoxyribose chain at the C1-position. When heated in the first thermal cycling step at the alkaline pH of Master Mix, the amplicon DNA chain breaks at the position of the deoxyuridine, thereby rendering the DNA non-amplifiable. AmpErase enzyme is inactive at temperatures above 55ºC, i.e., throughout the thermal cycling steps, and therefore does not destroy target amplicon. The cobas® CT/NG v2.0 Test has been demonstrated to inactivate at least 103 copies of deoxyuridine-containing CT/NG amplicon per PCR. Detection of PCR Products in the cobas® CT/NG v2.0 Test The cobas® CT/NG v2.0 Test utilizes real-time12,13 PCR technology. The use of fluorescent probes enables real-time detection of PCR product accumulation by monitoring the emission intensity of fluorescent dyes released during the amplification process. The probes include CT cryptic plasmid, CT ompA, NG DR-9A, NG DR-9B and CT/NG Internal Control-specific oligonucleotides, all labeled with a reporter dye and a quencher. When the fluorescent dye-labeled probes are intact, the reporter fluorescence is suppressed by the proximity of the quencher due to Förster-type energy transfer effects. During PCR, the probes hybridize to their respective target sequence and are cleaved by the 5΄ to 3΄ nuclease activity of the thermostable Z05 DNA polymerase. Once the reporter and quencher are separated, quenching no longer occurs, and the fluorescent emission of the reporter dyes increases. The amplification of CT targets, NG targets and the CT/NG Internal Control are measured independently and at different wavelengths. This process is repeated for a designated number of cycles, each cycle increasing the emission intensity of the individual reporter dyes. 9. Palmer, L. and Falkow, S. 1986. A common plasmid of Chlamydia trachomatis. Plasmid 16:52-63. 10. Peterson, E. M. and de la Maza, L.M. 1988, Restriction endonuclease analysis of DNA from Chlamydia trachomatis biovars. Journal of Clinical Microbiology 26:625-629. 11. Longo, M.C., Berninger, M.S. and Hartley, J.L. 1990. Use of uracil DNA glycosylase to control carry-over contamination in polymerase chain reactions. Gene. 93:125-128. 12. Higuchi, R., Dollinger, G., Walsh, P.S., and Griffith, R. 1992. Simultaneous amplification and detection of specific DNA sequences. Bio/Technology 10:413-417. 13. Heid, C.A., Stevens, J., Livak, J.K., and Williams, P.M. 1996. Real time quantitative PCR. Genome Research 6:986-994. en