Amvrosiadou Maria, Petropoulou Margarita, Poulou Myrto, Tzetis Maria, Kanavakis Emmanuel, Christopoulos Theodore K, Ioannou Penelope C
Laboratory of Analytical Chemistry, Department of Chemistry, Athens University, Athens 15771, Greece.
Laboratory of Medical Genetics, Athens University, Athens 11527, Greece.
J Chromatogr B Analyt Technol Biomed Life Sci. 2015 Dec 1;1006:201-208. doi: 10.1016/j.jchromb.2015.10.036. Epub 2015 Oct 30.
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.
威尔逊病是一种肝细胞铜转运的遗传性疾病，具有广泛的基因型和表型特征。ATP7B基因突变是导致该疾病的原因。迄今为止，已描述了ATP7B基因中约500种突变。我们报告了一种同时检测希腊患者中十种最常见ATP7B基因突变的方法。该方法包括3个简单步骤：（i）对突变侧翼的ATP7B基因片段进行多重PCR扩增；（ii）使用等位基因特异性引物对未纯化的扩增产物进行多重引物延伸反应；（iii）通过使用抗生物素缀合金纳米颗粒的干试剂多等位基因试纸条测定法在数分钟内目视检测引物延伸反应产物。对PEXT反应的效率和特异性进行了优化研究。通过对46个已知基因型的DNA样本和34个盲样进行基因分型来评估该方法。结果与参考方法获得的结果完全一致。该方法简单、快速、经济高效，不需要专门的仪器或高素质的人员。
