Al-Madhagi Sallam, Joda Hamdi, Jauset-Rubio Miriam, Ortiz Mayreli, Katakis Ioanis, O'Sullivan Ciara K
Interfibio Research Group, Department of Chemical Engineering, Universitat Rovira i Virgili, Avinguda Països Catalans 26, 43007, Tarragona, Spain.
Interfibio Research Group, Department of Chemical Engineering, Universitat Rovira i Virgili, Avinguda Països Catalans 26, 43007, Tarragona, Spain.
Anal Biochem. 2018 Sep 1;556:16-22. doi: 10.1016/j.ab.2018.06.013. Epub 2018 Jun 18.
DNA biosensors are attractive tools for genetic analysis as there is an increasing need for rapid and low-cost DNA analysis, primarily driven by applications in personalized pharmacogenomics, clinical diagnostics, rapid pathogen detection, food traceability and forensics. A rapid electrochemical genosensor detection methodology exploiting a combination of modified primers for solution-phase isothermal amplification, followed by rapid detection via hybridization on gold electrodes is reported. Modified reverse primers, exploiting a C18 spacer between the primer-binding site and an engineered single stranded tail, are used in a recombinase polymerase amplification reaction to produce an amplicon with a central duplex flanked by two single stranded tails. These tails are designed to be complementary to a gold electrode tethered capture oligo probe as well as a horseradish peroxidase labelled reporter oligo probe. The time required for hybridization of the isothermally generated amplicons with each of the immobilized and reporter probes was optimised to be 2 min, in both cases. The effect of amplification time and the limit of detection were evaluated using these hybridization times for both single stranded and double stranded DNA templates. The best detection limit of 70 fM for a ssDNA template was achieved using 45 min amplification, whilst for a dsDNA template, just 30 min amplification resulted in a slightly lower detection limit of 14 fM, whilst both 20 and 45 min amplification times were observed to provide detection limits of 71 and 72 fM, respectively, but 30 and 45 min amplification resulted in a much higher signal and sensitivity. The genosensor was applied to genomic DNA and real patient and control blood samples for detection of the coeliac disease associated DQB1*02 HLA allele, as a model system, demonstrating the possibility to carry out molecular diagnostics, combining amplification and detection in a rapid and facile manner.
DNA生物传感器是用于基因分析的有吸引力的工具，因为对快速且低成本的DNA分析的需求日益增加，这主要是由个性化药物基因组学、临床诊断、快速病原体检测、食品可追溯性和法医学中的应用所推动的。本文报道了一种快速电化学基因传感器检测方法，该方法利用修饰引物进行溶液相等温扩增，然后通过在金电极上杂交进行快速检测。修饰的反向引物在引物结合位点和工程化单链尾之间利用C18间隔区，用于重组酶聚合酶扩增反应，以产生具有中央双链体且两侧为两个单链尾的扩增子。这些尾被设计为与金电极固定的捕获寡核苷酸探针以及辣根过氧化物酶标记的报告寡核苷酸探针互补。在两种情况下，等温生成的扩增子与固定化探针和报告探针各自杂交所需的时间均优化为2分钟。使用这些杂交时间评估了单链和双链DNA模板的扩增时间和检测限的影响。对于单链DNA模板，使用45分钟扩增可实现70 fM的最佳检测限，而对于双链DNA模板，仅30分钟扩增就产生了略低的14 fM检测限，同时观察到20分钟和45分钟的扩增时间分别提供71和72 fM的检测限，但30分钟和45分钟的扩增产生了更高的信号和灵敏度。作为模型系统，该基因传感器应用于基因组DNA以及真实患者和对照血样，以检测与乳糜泻相关的DQB1*02 HLA等位基因，证明了以快速简便的方式结合扩增和检测进行分子诊断的可能性。
