For in vitro diagnostic use. Others cobas HSV 1 and 2 Test IVD cobas® HSV 1 and 2 Test RMD-4800-HSV-001 for use on the cobas® 4800 system 06 768 199 190 6 768 199 190 06768199190 6768199190 06768199190 KIT COBAS 4800 HSV 1&2 AMP/DET 80T IVD cobas HSV 1 and 2 Test 00875197005226 Reagents, kits 1 kit 80 tests true The cobas® HSV 1 and 2 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 and typing of Herpes simplex virus 1 and 2 (HSV-1 and HSV-2) DNA in clinician-collected anogenital lesion specimens from symptomatic male and female patients. The cobas® HSV 1 and 2 Test is intended for use as an aid in diagnosis of anogenital HSV-1 and HSV-2 infections in symptomatic patients. en The cobas® HSV 1 and 2 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 and differentiation of Herpes simplex virus 1 and 2 (HSV-1 and HSV-2) DNA in clinician-collected, external anogenital lesion specimens from symptomatic male and female patients. The cobas® HSV 1 and 2 Test is intended for use as an aid in diagnosis of anogenital HSV-1 and HSV-2 infections in symptomatic patients.Warning: The cobas® HSV 1 and 2 Test is not FDA cleared for use with cerebrospinal fluid (CSF) and is not intended to be used for prenatal screening or for individuals under the age of 18 years. en Sample PreparationSample preparation for the cobas® HSV 1 and 2 Test is automated with the use of the cobas® x 480 instrument. Viruses in the anogenital lesion samples 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® DetectionThe 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 five targets: the DNA polymerase region B and Thymidine Kinase region C of HSV-1; the Glycoprotein B 3’ end region and Thymidine Kinase region C of HSV-2, and Internal Control. The dual target design for HSV-1 and HSV-2 enhances the assay robustness. If the targets 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 AmplificationSelective amplification of target nucleic acid from the specimen is achieved in the cobas® HSV 1 and 2 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 deoxyuridine13, 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® HSV 1 and 2 Test has been demonstrated to inactivate at least 1000 copies of deoxyuridine-containing HSV 1 and 2 amplicon per PCR.13. Longo MC, Berninger MS, Hartley JL. Use of uracil DNA glycosylase to control carry-over contamination in polymerase chain reactions. Gene. 1990;93(1):125-128. en The cobas® HSV 1 and 2Test contains two major processes: (1) automated sample preparation to extract nucleic acids from the anogenitallesion specimens; (2) PCR amplification of target DNA sequences using HSV-1 and HSV-2 specific primers, and real-time detection of cleaved fluorescent-labeled HSV-1and HSV-2 specific oligonucleotide detection probes. An Internal Control, containing unrelated randomized DNA sequence, 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 preparationSample preparation for the cobas® HSV 1 and 2 Test is automated with the use of the cobas® x 480 instrument. Viruses in the anogenital lesion samples 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® detectionThe 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 five targets: the DNA polymerase region B and Thymidine Kinase region C of HSV-1 gene; the Glycoprotein B 3’ end region and Thymidine Kinase region C of HSV-2 gene, and Internal Control. The dual target design for HSV-1 and HSV-2 enhances the assay robustness. If the targets 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 amplificationSelective amplification of target nucleic acid from the specimen is achieved in the cobas® HSV 1 and 2 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 deoxyuridine10, 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® HSV 1 and 2 Test has been demonstrated to inactivate at least 1000 copies of deoxyuridine-containing HSV 1 and 2 amplicon per PCR.10. Longo MC, Berninger MS, Hartley JL. Use of uracil DNA glycosylase to control carry-over contamination in polymerase chain reactions. Gene. 1990;93(1):125-128. en

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