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Τwo-panel molecular testing for genetic predisposition for thrombosis using multi-allele visual biosensors

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Abstract

Thrombosis is considered as the most typical example of multigenic/multifactorial disorder. The three most common genetic risk factors for thrombotic disorders are the G1691A mutation in factor V gene (FV Leiden), the G20210Α mutation in prothrombin gene (FII), and the C677T mutation in the methylenetetrahydrofolate reductase (MTHFR) gene. An additional panel of biomarkers predisposing for thrombotic events includes the H1299R variant in factor V gene (HR2), A1298C variant in MTHFR gene, the V34L mutation in fibrinogen stabilizing factor XIII (FXIII) gene as well as the 4G/5G polymorphism in plasminogen activator inhibitor type-1 (PAI-1) gene. In this context, we report a novel, rapid and low-cost two-panel diagnostic platform for the simultaneous visual genotyping of the seven mutations (14 alleles). The proposed method comprises the following: (a) a multiplex PCR using genomic DNA isolated from peripheral blood, (b) a multiplex genotyping reaction based on allele-specific primer extension, and (c) visual detection of the genotyping reaction products by means of a multi-allele dipstick-type DNA biosensor, using gold nanoparticles as reporters. The method was applied to 40, previously characterized, and 15 blind clinical samples and the results were 100 % accurate. The proposed assay is simple to perform, requires no specialized and costly equipment, and eliminates multiple pipetting, incubation, and washing steps.

Genotyping platform for visual detection of point mutations using PEXT reaction and multi-allele DNA biosensor

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Acknowledgments

We would like to thank Dr. Georgios Vartholomatos from the Hematology Laboratory-Molecular Biology Unit of the Ioannina University Hospital, Greece, for providing the clinical samples.

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    Authors and Affiliations

    1. Laboratory of Analytical Chemistry, Department of Chemistry, Athens University, Athens, 15771, Greece

      Nikolaos Fountoglou, Margarita Petropoulou, Alexandra Iliadi & Penelope C. Ioannou

    2. Department of Chemistry, University of Patras, Patras, 26500, Greece

      Theodore K. Christopoulos

    3. Foundation for Research and Technology Hellas, Institute of Chemical Engineering and High Temperature Chemical Processes (FORTH/ICE-HT), Patras, 26504, Greece

      Theodore K. Christopoulos

    Authors
    1. Nikolaos Fountoglou
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    2. Margarita Petropoulou
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    3. Alexandra Iliadi
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    4. Theodore K. Christopoulos
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    5. Penelope C. Ioannou
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    Correspondence to Penelope C. Ioannou.

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    Nikolaos Fountoglou and Margarita Petropoulou contributed equally to this work.

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    Fountoglou, N., Petropoulou, M., Iliadi, A. et al. Τwo-panel molecular testing for genetic predisposition for thrombosis using multi-allele visual biosensors. Anal Bioanal Chem 408, 1943–1952 (2016). https://doi.org/10.1007/s00216-016-9310-9

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    • DOI: https://doi.org/10.1007/s00216-016-9310-9

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