AB-THROMBO TYPE PLUS code 04-71A-20 - 1 Pages

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pANALITICA Advanced biomedicine www.abanalitica.it AB-THROMBO TYPE PLUS code 04-71A-20 Kit for simultaneous detection of mutations in the genes coding for Factor II, Factor V, MTHFR and PAI-1 by reverse line blot Number of tests: 20 Shelf life: 6 months Sample material: DNA extract Amplification: Ready-to-use single-dose premix format Detected mutations: C€ Ovd Vein thrombosis is the impediment of blood circulation caused by blood clots that may have formed in the same vein or may have originated elsewhere in the body. Although, blood clots most commonly form in the superficial and deep veins of the legs they can also be released in the veins of the brain, the retina, the liver or the mesentery. Activation of the coagulation system and clot formation can be facilitated by external influences like trauma, surgery, immobilization, pregnancy and the use of oral contraceptives as well as by the individual genetic background. Mutations in factors that are involved in hemostasis and fibrinolysis may bring with them a permanently elevated susceptibility for venous thrombosis. To date, numerous mutations have been identified that increase the susceptibility for venous thrombosis. Among these are mutations in the genes coding for coagulation Factor V, coagulation Factor II, the methylene-tetrahydrofolate reductase (MTHFR) and    the plasminogen activator inhibitor-1 (PAI-1). Since it has been reported that the presence of multiple mutations may have synergistic effects, an assay that allows simultaneous detection of the most common mutations is of great value for clinicians. TEST PRINCIPLE GENEQUALITY AB-THROMBO TYPE PLUS is an IVD for the simultaneous detection and identification of the most common mutations related to thrombosis. The kit is based on PCR and reverse allele specific hybridization (reverse line blot). The following mutations can be detected: Factor II G20210A, FactorV Leiden (G1691A, Arg506Gln), Factor VH1299R (HR2 haplotype), MTHFR C677T, MTHFR A1298C and the PAI-1 4G/5G polymorphism. Staining control Factor II WT Factor II MUT Factor V Leiden WT Factor V Leiden MUT Factor V HR2 WT Factor V HR2 MUT Accuracy: 100% Duration of protocol: approx. 4 hours TEST PROCEDURE 1 DNA AMPLIFICATION: Multiplex PCR in single-dose vials after addition of oligomix (approx. 1.5 h) 2 SAMPLE DENATURATION: Incubation with denatur-ation solution for 5 min at room temperature 3 HYBRIDIZATION: Incubation of denatured biotinylated PCR product with strip at 46 °C for 30 min 4 COLORIMETRIC DETECTION: Incubation with strept-avidin at 46 °C for 30 minutes, and staining of strips with chromogenic NBT/BCIP for 10 min REFERENCES Cover the developed strip with the Reading card (transparent film for strip interpretation) to quickly and easily identify whether and which mutations are present and assess the genotype (homozygous, heterozygous, compound heterozygous). Bertina RM, etal., Nature 1994; 369:64-67. Gemmati D, et al., ArteriosclerThromb Vase Biol. 1999; 19: 1761-7 Meinkoth, J. and Wahl, G. In Analytical Biochemistry, 138: 267, 1984. Poort SR, Rosendaal FR, Reitsma PH, Bertina RM, Blood 1996; 88:3698-3703. AB ANALITICA srl Via Svizzera 16 - 35127 PADUA (ITALY) tel. +39 049 761698 e-mail: info@abanalitica.it www.abanalitica.it

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