For in vitro diagnostic use. Others cobas Factor II and Factor V Test IVD cobas® Factor II and Factor V Test RMD-4800-F2F5-001 07 948 352 190 7 948 352 190 07948352190 7948352190 07948352190 KIT COBAS 4800 F2F5 AMP/DET 96T IVD cobas Factor II and Factor V Test 00875197005998 Reagents, kits 1 kit 96 tests true The cobas® Factor II and Factor V Test is an in vitro diagnostic device that uses real-time Polymerase Chain Reaction (PCR) for the detection and genotyping of the human Factor II (Prothrombin) G20210A mutation and the human Factor V Leiden G1691A mutation, from genomic DNA obtained from K2EDTA whole blood specimens, as an aid in diagnosis of patients with suspected thrombophilia. The cobas® Factor II and Factor V Test and the cobas z 480 analyzer are used together for automated amplification and detection. en The cobas® Factor II and Factor V Test is an in vitro diagnostic device that uses real-time Polymerase Chain Reaction (PCR) for the detection and genotyping of the human Factor II (Prothrombin) G20210A mutation and the human Factor V Leiden mutation, from genomic DNA obtained from K2EDTA whole blood specimens, as an aid in diagnosis of patients with suspected thrombophilia. The cobas® Factor II and Factor V Test and the cobas z 480 analyzer are used together for automated amplification and detection. en The cobas® Factor II and Factor V Test uses real-time PCR analysis of genomic DNA samples isolated from K2EDTA whole blood to determine the genotype of the Factor II gene at position 20210 and/or the genotype of the Factor V gene at position 1691. The test is performed on the cobas z 480 analyzer. A positive control (F2F5 PC) and negative control (F2F5 NC) are included in each run to confirm the validity of the run.Sample preparationIsolation of genomic DNA from K2EDTA whole blood specimens may be achieved using DNA isolation methods that yield DNA of sufficient concentration (≥ 0.1 ng/μL).PCR amplificationTarget selectionThe cobas® Factor II and Factor V Test contains two PCR primer pairs that amplify a 173 base pair region of the Factor II gene and a 233 base pair region of the Factor V gene that contain the sites of the Factor II (prothrombin) and Factor V Leiden mutations, respectively. Both wild type and mutant alleles are amplified for each gene.Target amplificationThermus species Z05 DNA polymerase and PCR primers are used for amplification of the Factor II and Factor V target regions. First, the PCR mixture is heated to denature the genomic DNA and expose the primer target sequences. As the mixture cools, the upstream and downstream primers anneal to the target DNA sequences in the Factor II and Factor V genes, and Z05 DNA polymerase, in the presence of divalent metal cation and excess deoxynucleotide triphosphates (dNTPs), extends each annealed primer using the target DNA as a template. This completes the first cycle of PCR, yielding double-stranded DNA copies (amplicons) of the Factor II target region and the Factor V target region. This process is repeated for a number of cycles, with each cycle effectively doubling the amount of Factor II and Factor V DNA for the target regions.Automated real-time genotypingThe cobas® Factor II and Factor V Test utilizes real-time PCR technology to determine the genotype of the Factor II gene at position 20210 and the genotype of the Factor V gene at position 1691. The reaction contains four oligonucleotide probes, two for Factor II (G20210A mutation and wild type sequences), and two for Factor V (G1691A mutation and wild type sequences). Each oligonucleotide probe is labeled with a fluorescent dye that serves as a reporter, and with a quencher molecule that absorbs (quenches) fluorescent emissions from the reporter dye within an intact probe. During each cycle of amplification, the probes bind to the complementary regions of amplicon DNA, and are cleaved by the 5’ to 3’ nuclease activity of Z05 DNA Polymerase. The wild type and mutant probes are cleaved only when they are bound to the corresponding wild type or mutant Factor II and Factor V sequences, respectively.Cleavage of probe molecules allows the reporter dye to separate from the quencher, so that the fluorescence of the reporter dye can be measured when the reaction is irradiated with the appropriate wavelength of light. The Factor II and Factor V wild type and mutant probes are labeled with different reporter dyes. Detection of elevated fluorescence for any of the reporter dyes during real-time PCR indicates that the Factor II or Factor V allele corresponding to that dye is present in the reaction. The Factor II and Factor V genotypes are determined based on which alleles are detected for each gene. If both mutant and wild type alleles are detected for a gene, the genotype is heterozygous. If only the wild type or mutant alleles are detected, then the genotype is wild type or homozygous mutant, respectively. If neither the wild type nor the mutant allele is detected for either Factor II or Factor V, the result for both genes is invalid.Selective amplificationSelective amplification of target nucleic acid from the sample is achieved in the cobas® Factor II and Factor V Test by the use of AmpErase (uracil-N-glycosylase enzyme) and deoxyuridine triphosphate (dUTP).8 The AmpErase enzyme recognizes and catalyzes the destruction of DNA strands containing deoxyuridine but not DNA containing thymidine. Deoxyuridine is not present in naturally occurring DNA but is always present in amplicon DNA due to the use of dUTP in place of deoxythymidine triphosphate as one of the deoxynucleotide triphosphates in the Master Mix reagent; therefore, only amplicon DNA contains deoxyuridine. Deoxyuridine renders contaminating amplicon susceptible to destruction by AmpErase enzyme prior to amplification of the target DNA. The 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 alkaline pH, the amplicon DNA chain breaks at the position of the deoxyuridine, thereby rendering the DNA non-amplifiable. The AmpErase enzyme is inactive at temperatures above 55ºC, i.e., throughout the thermal cycling steps, and therefore does not destroy target amplicon.8. Longo MC, Berninger MS, Hartley JL. Use of uracil DNA glycosylase to control carry-over contaminationin polymerase chain reactions. Gene. 1990;93:125-128. en The cobas® Factor II and Factor V Test uses real-time PCR analysis of genomic DNA samples isolated from K2EDTA whole blood to determine the genotype of the Factor II gene at position 20210 and/or the genotype of the Factor V gene at position 1691. The test is performed on the cobas z 480 analyzer. A positive control (F2F5 PC) and negative control (F2F5 NC) are included in each run to confirm the validity of the run.Sample preparationIsolation of genomic DNA from K2EDTA whole blood specimens may be achieved using DNA isolation methods that yield DNA of sufficient concentration (≥ 0.1 ng/μL).PCR amplificationTarget selectionThe cobas® Factor II and Factor V Test contains two PCR primer pairs that amplify a 173 base pair region of the Factor II gene and a 233 base pair region of the Factor V gene that contain the sites of the Factor II (prothrombin) and Factor V Leiden mutations, respectively. Both wild type and mutant alleles are amplified for each gene.Target amplificationThermus species Z05 DNA polymerase and PCR primers are used for amplification of the Factor II and Factor V target regions. First, the PCR mixture is heated to denature the genomic DNA and expose the primer target sequences. As the mixture cools, the upstream and downstream primers anneal to the target DNA sequences in the Factor II and Factor V genes, and Z05 DNA polymerase, in the presence of divalent metal cation and excess dinucleotide triphosphates (dNTPs), extends each annealed primer using the target DNA as a template. This completes the first cycle of PCR, yielding double-stranded DNA copies (amplicons) of the Factor II target region and the Factor V target region. This process is repeated for a number of cycles, with each cycle effectively doubling the amount of Factor II and Factor V DNA for the target regions.Automated real-time genotypingThe cobas® Factor II and Factor V Test utilizes real-time PCR technology to determine the genotype of the Factor II gene at position 20210 and the genotype of the Factor V gene at position 1691. The reaction contains four oligonucleotide probes, two for Factor II (G20210A mutation and wild type sequences), and two for Factor V (G1691A mutation and wild type sequences). Each oligonucleotide probe is labeled with a fluorescent dye that serves as a reporter, and with a quencher molecule that absorbs (quenches) fluorescent emissions from the reporter dye within an intact probe. During each cycle of amplification, the probes bind to the complementary regions of amplicon DNA, and are cleaved by the 5’ to 3’ nuclease activity of Z05 DNA Polymerase. The wild type and mutant probes are cleaved only when they are bound to the corresponding wild type or mutant Factor II and Factor V sequences, respectively.Cleavage of probe molecules allows the reporter dye to separate from the quencher, so that the fluorescence of the reporter dye can be measured when the reaction is irradiated with the appropriate wavelength of light. The Factor II and Factor V wild type and mutant probes are labeled with different reporter dyes. Detection of elevated fluorescence for any of the reporter dyes during real-time PCR indicates that the Factor II or Factor V allele corresponding to that dye is present in the reaction. The Factor II and Factor V genotypes are determined based on which alleles are detected for each gene. If both mutant and wild type alleles are detected for a gene, the genotype is heterozygous. If only the wild type or mutant alleles are detected, then the genotype is wild type or homozygous mutant, respectively. If neither the wild type nor the mutant allele is detected for either Factor II or Factor V, the result for both genes is invalid.Selective amplificationSelective amplification of target nucleic acid from the sample is achieved in the cobas® Factor II and Factor V Test by the use of AmpErase (uracil-N-glycosylase enzyme) and deoxyuridine triphosphate (dUTP).8 The AmpErase enzyme recognizes and catalyzes the destruction of DNA strands containing deoxyuridine but not DNA containing thymidine. Deoxyuridine is not present in naturally occurring DNA but is always present in amplicon DNA due to the use of dUTP in place of deoxythymidine triphosphate as one of the dinucleotide triphosphates in the Master Mix reagent; therefore, only amplicon DNA contains deoxyuridine. Deoxyuridine renders contaminating amplicon susceptible to destruction by AmpErase enzyme prior to amplification of the target DNA. The 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 alkaline pH, the amplicon DNA chain breaks at the position of the deoxyuridine, thereby rendering the DNA non-amplifiable. The AmpErase enzyme is inactive at temperatures above 55ºC, i.e., throughout the thermal cycling steps, and therefore does not destroy target amplicon.8. Longo MC, Berninger MS, Hartley JL. Use of uracil DNA glycosylase to control carry-over contaminationin polymerase chain reactions. Gene. 1990;93:125-128. en

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