title: Τwo-panel molecular testing for genetic predisposition for thrombosis using multi-allele visual biosensors.
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.
authors.affiliation: Laboratory of Analytical Chemistry, Department of Chemistry, Athens University, Athens, 15771, Greece.
authors.forename: Nikolaos
authors.initials: N
authors.lastname: Fountoglou
authors.affiliation: Laboratory of Analytical Chemistry, Department of Chemistry, Athens University, Athens, 15771, Greece.
authors.forename: Margarita
authors.initials: M
authors.lastname: Petropoulou
authors.affiliation: Laboratory of Analytical Chemistry, Department of Chemistry, Athens University, Athens, 15771, Greece.
authors.forename: Alexandra
authors.initials: A
authors.lastname: Iliadi
authors.affiliation: Department of Chemistry, University of Patras, Patras, 26500, Greece.|Foundation for Research and Technology Hellas, Institute of Chemical Engineering and High Temperature Chemical Processes (FORTH/ICE-HT), Patras, 26504, Greece.
authors.forename: Theodore K
authors.initials: TK
authors.lastname: Christopoulos
authors.affiliation: Laboratory of Analytical Chemistry, Department of Chemistry, Athens University, Athens, 15771, Greece. ioannou@chem.uoa.gr.
authors.forename: Penelope C
authors.initials: PC
authors.lastname: Ioannou
mesh_terms: D000483:Alleles; D015374:Biosensing Techniques / Q000379:methods*; D004247:DNA / Q000235:genetics*; D004252:DNA Mutational Analysis / Q000379:methods*; D005165:Factor V / Q000235:genetics; D005176:Factor XIII / Q000235:genetics; D020022:Genetic Predisposition to Disease; D060005:Genotyping Techniques / Q000379:methods*; D006801:Humans; D042965:Methylenetetrahydrofolate Reductase (NADPH2) / Q000235:genetics; D017395:Plasminogen Activator Inhibitor 1 / Q000235:genetics; D017354:Point Mutation; D016133:Polymerase Chain Reaction / Q000379:methods; D011110:Polymorphism, Genetic; D011516:Prothrombin / Q000235:genetics; D013927:Thrombosis / Q000235:genetics*
chemical_list: D017395:Plasminogen Activator Inhibitor 1; C095381:factor V Leiden; D005165:Factor V; D011516:Prothrombin; D004247:DNA; D005176:Factor XIII; C573423:MTHFR protein, human; D042965:Methylenetetrahydrofolate Reductase (NADPH2)
keywords: Molecular testing; Multi-allele DNA biosensor; Primer extension reaction; Thrombophilia