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  • Detection of the Factor V L...
    Orum, Henrik; Jakobsen, Mogens H; Koch, Troels; Vuust, Jens; Borre, Martin B

    Clinical chemistry (Baltimore, Md.), 11/1999, Letnik: 45, Številka: 11
    Journal Article, Conference Proceeding

    Individuals carrying the factor V Leiden mutation have been shown to have an increased risk of developing venous thromboembolism. Our aim was to develop an ELISA-like assay to detect the mutation in PCR-amplified genomic DNA using novel, high-affinity DNA analogs, termed locked nucleic acids (LNAs). LNA octamer probes complementary to the factor V wild-type or mutated sequence were covalently attached to individual wells of a microtiter plate. Biotinylated factor V amplicons were added, and hybridization to the immobilized LNA probes was scored colorimetrically using a horseradish peroxidase-anti-biotin Fab conjugate and tetramethylbenzidine substrate. In a prospective study of 53 patients, the assay reproducibly scored both factor V homozygotes and heterozygotes with excellent sensitivity and specificity. All results were in complete agreement with the results obtained with the conventional PCR-restriction fragment length polymorphism technique. The simplicity of the assay and its procedural relatedness to the widely used ELISA format should make it useful for routine factor V testing in the clinical laboratory.