Multi-allele genotyping platform for the simultaneous detection of mutations in the Wilson disease related ATP7B gene

作者：Amvrosiadou Maria; Petropoulou Margarita; Poulou Myrto; Tzetis Maria; Kanavakis Emmanuel; Christopoulos Theodore K; Ioannou Penelope C^*

来源：Journal of Chromatography B-Analytical Technologies in the Biomedical and Life Sciences, 2015, 1006: 201-208.

DOI：10.1016/j.jchromb.2015.10.036

• Wilson disease
• ATP7B gene mutations
• Genotyping
• Primer extension reaction
• Visual detection
• Multi-allele DNA biosensor

Wilson's disease is an inherited disorder of copper transport in the hepatocytes with a wide range of genotype and phenotype characteristics. Mutations in the ATP7B gene are responsible for the disease. Approximately, over 500 mutations in the ATP7B gene have been described to date. We report a method for the simultaneous detection of the ten most common ATP7B gene mutations in Greek patients. The method comprises 3 simple steps: (i) multiplex PCR amplification of fragments in the ATP7B gene flanking the mutations (ii) multiplex primer extension reaction of the unpurified amplification products using allele-specific primers and (iii) visual detection of the primer extension reaction products within minutes by means of dry-reagent multi-allele dipstick assay using anti-biotin conjugated gold nanoparticles. Optimization studies on the efficiency and specificity of the PEXT reaction were performed. The method was evaluated by genotyping 46 DNA samples of known genotype and 34 blind samples. The results were fully concordant with those obtained by reference methods. The method is simple, rapid, cost-effective and it does not require specialized instrumentation or highly qualified personnel.

• 出版日期2015-12-1