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

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• Thrombosis is a complex disorder influenced by multiple genetic factors.
• Key genetic risk factors include mutations in Factor V (FV Leiden, HR2), Prothrombin (FII), Methylenetetrahydrofolate Reductase (MTHFR C677T, A1298C), Factor XIII (FXIII V34L), and PAI-1 (4G/5G).
• Accurate and efficient identification of these genetic predispositions is crucial for managing thrombotic risk.

Purpose of the Study:

• To develop and validate a novel, rapid, and cost-effective diagnostic platform.
• To enable simultaneous visual genotyping of seven common mutations associated with thrombotic disorders.
• To provide a simplified and accessible tool for clinical genetic testing.

Main Methods:

• A multiplex PCR amplification of genomic DNA from peripheral blood.
• A multiplex genotyping reaction utilizing allele-specific primer extension.
• Visual detection of amplified products using a multi-allele dipstick DNA biosensor with gold nanoparticle reporters.

Main Results:

• The developed platform successfully performed simultaneous visual genotyping of all seven target mutations (14 alleles).
• The assay demonstrated 100% accuracy when tested on 40 characterized and 15 blind clinical samples.
• The method is simple, requires no specialized equipment, and reduces procedural steps.

Conclusions:

• The novel diagnostic platform offers a rapid, accurate, and low-cost solution for genotyping multiple thrombotic risk factors.
• This visual, dipstick-based assay simplifies genetic testing for inherited thrombophilia.
• The technology has the potential to improve the accessibility and efficiency of thrombotic risk assessment in clinical settings.