We developed a rapid and low-cost panel of three assays for visual genotyping of the three most common genetic risk factors in thrombophilia, namely, the single-point mutations in the FV (Leiden factor), FII and MTHFR genes. A triplex PCR was developed for simultaneous amplification of three fragments spanning the interrogated loci. Allele discrimination was accomplished by a 5-min primer extension reaction on each locus. Detection of the extension products was performed by means of a dual-allele dipstick-type DNA biosensor. The biosensor is disposable and allows visual detection and confirmation of the genotyping products within 15 min by hybridization without the need for specialized instruments or expensive reagents. The proposed method was evaluated by genotyping 40 samples with a variety of genotypes for all three mutations. The genotyping results were in full concordance with those obtained by restriction fragment length polymorphism analysis and sequencing.
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