For in vitro diagnostic use. Others cobas Cdiff Test IVD cobas® Cdiff Test RMD-4800-CDIFF-001 for use on the cobas® 4800 system 06 768 237 190 6 768 237 190 06768237190 6768237190 06768237190 KIT COBAS 4800 CDIFF AMP/DET 80T IVD cobas Cdiff Test 00875197005240 Reagents, kits 1 kit 80 tests true The cobas® Cdiff Test on the cobas® 4800 System is an automated, qualitative in vitro diagnostic test, that utilizes real-time polymerase chain reaction (PCR), for the direct detection of the toxin B (tcdB) gene of toxigenic Clostridium difficile (C. difficile) in unformed (liquid or soft) stool specimens obtained from patients suspected of having C. difficile infection (CDI). The cobas® Cdiff Test is intended for use as an aid in the diagnosis of CDI in humans in conjunction with clinical and epidemiological risk factors. en Sample preparation Sample preparation for the cobas® Cdiff Test is automated with the use of the cobas® x 480 instrument. Organisms are lysed with chaotropic agent, proteinase K, and SDS reagents. Released nucleic acids, along with added Internal Control DNA, are bound by magnetic glass particles. They are washed and then eluted into a small volume of buffer. The instrument then takes an aliquot of the eluted material and sets up the PCR reaction with an activated Master Mix. PCR amplification and TaqMan® detection The PCR cycling steps and detection of target signal occurs in the cobas® z 480 analyzer. The Master Mix reagent contains primer pairs and probes for two targets: toxin B and Internal Control. If the target nucleic acid sequences are present, amplification with the corresponding primers will occur by a thermostable DNA polymerase, generating PCR products (amplicon). These products are detected by specific TaqMan probes containing a fluorescent dye and a quencher. Normally, the quencher suppresses the fluorescence of the dye. However, if the PCR product is present, the probe hybridizes to the product and gets cleaved by the 5’ to 3’ nuclease activity of the polymerase. This reaction allows the fluorescence to be emitted from the dye, and the signal is recorded in real time during each PCR cycle by the cobas® z 480 analyzer. The signal is interpreted by the cobas® 4800 System Software and reported as final results. Selective amplification Selective amplification of target nucleic acid from the specimen is achieved in the cobas® Cdiff Test by the use of AmpErase (uracil-N-glycosylase) enzyme and deoxyuridine triphosphate (dUTP). The AmpErase enzyme recognizes and catalyzes the destruction of DNA strands containing deoxyuridine23, 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® Cdiff Test has been demonstrated to inactivate at least 1000 copies of deoxyuridine-containing C. difficile amplicon per PCR. 23. Hardy K, Price C, Szczepura A, et al. Reduction in the rate of methicillin-resistant Staphylococcus aureus acquisition in surgical wards by rapid screening for colonization: a prospective, cross-over study. Clin Microbiol Infect. 2010;16(4):333-339. Epub 2009/07/23. en Sample preparation Sample preparation for the cobas® Cdiff Test is automated with the use of the cobas® x 480 instrument. Organismsare lysed with chaotropic agent, proteinase K, and SDS reagents. Released nucleic acids, along with added Internal Control DNA, are bound by magnetic glass particles. They are washed and then eluted into a small volume of buffer. The instrument then takes an aliquot of the eluted material and sets up the PCR reaction with an activated Master Mix. PCRamplification and TaqMan® detection The PCR cycling steps and detection of target signal occurs in the cobas® z 480 analyzer. The Master Mix reagent contains primer pairs and probes for two targets: toxin B and Internal Control. If the target nucleic acid sequences are present, amplification with the corresponding primers will occur by a thermostable DNA polymerase, generating PCR products (amplicons). These products are detected by specific TaqMan probes containing a fluorescent dye and a quencher. Normally, the quencher suppresses the fluorescence of the dye. However, if the PCR product is present, the probe hybridizes to the product and gets cleaved by the 5’ to 3’ nuclease activity of the polymerase. This reaction allows the fluorescence to be emitted from the dye, and the signal is recorded in real time during each PCR cycle by the cobas® z 480 analyzer. The signal is interpreted by the cobas® 4800 System Software and reported as final results. Selective amplification Selective amplification of target nucleic acid from the specimen is achieved in the cobas® Cdiff Test by the use of AmpErase (uracil-N-glycosylase) enzyme and deoxyuridine triphosphate (dUTP). The AmpErase enzyme recognizes and catalyzes the destruction of DNA strands containing deoxyuridine,23 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 openingthe 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® Cdiff Test has been demonstrated to inactivate at least 1000 copies of deoxyuridine-containing C. difficile amplicon per PCR. 23. Hardy K, Price C, Szczepura A, et al. Reduction in the rate of methicillin-resistant Staphylococcus aureus acquisition in surgical wards by rapid screening for colonization: a prospective, cross-over study. Clin Microbiol Infect. 2010;16(4):333-339. Epub 2009/07/23. en

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