School of Life Sciences, Anhui Medical University, Hefei, Anhui, 230032, PR China.
Engineering Research Center of Bio-process, MOE, School of Food and Biological Engineering, Hefei University of Technology, Hefei, 230009, PR China.
Anal Chim Acta. 2022 Oct 16;1230:340421. doi: 10.1016/j.aca.2022.340421. Epub 2022 Sep 21.
Single base mutations detection is crucial for the diagnosis and treatment of cancer. However, the current methods with poor selectivity and sensitivity required large instruments, which are difficult to meet clinical demands. Herein, we develop a CRISPR/Cas9 based visual colorimetric platform to specifically detect all single base mutations. In this strategy, the Recombinase Polymerase Amplification (RPA) was firstly used to amplify the target, and introduced the PAM site in the target DNA sequence by designing the point mutation primer, thus achieving detection for all single base mutations by the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated specific recognition. With the help of CRISPR/Cas9 system, those RPA products can release single strand DNA to hybridize with the padlock probe and trigger rolling circle amplification (RCA). Based on the magnetic separation, HRP-gold nanoparticles complex (hGNPs) and biotin modified probe (Bio-probe) were further used to achieve enhanced visual variations assay by hybridizing with RCA products. Benefiting from the RPA assisted triple signal amplification, this method not only showed enhanced sensitivity with a limit of detection (LOD) as low as 0.2 fM and 0.01% of KRAS-G12D mutation percentage, but the specificity against KRAS-G12D mutation also be synergistically enhanced by combining the CRISPR/Cas9-mediated specific recognition with the specific T4 ligation reaction of RCA system. Furthermore, this system has been successfully used to visually detect genome in serum, suggesting its great potential for point-of-care diagnosis in clinical.
单碱基突变检测对于癌症的诊断和治疗至关重要。然而，目前的方法选择性和灵敏度差，需要大型仪器，难以满足临床需求。在此，我们开发了一种基于 CRISPR/Cas9 的可视化比色平台，用于特异性检测所有单碱基突变。在该策略中，首先使用重组酶聚合酶扩增（RPA）扩增靶标，并通过设计点突变引物在靶标 DNA 序列中引入 PAM 位点，从而通过 CRISPR/Cas9 介导的特异性识别实现所有单碱基突变的检测。借助 CRISPR/Cas9 系统，这些 RPA 产物可以释放单链 DNA 与发夹探针杂交，并触发滚环扩增（RCA）。基于磁分离，进一步使用 HRP-金纳米粒子复合物（hGNPs）和生物素修饰探针（Bio-probe）与 RCA 产物杂交，实现增强的可视化变化检测。受益于 RPA 辅助的三重信号放大，该方法不仅显示出低至 0.2 fM 的检测限（LOD）和 0.01%的 KRAS-G12D 突变百分比的增强灵敏度，而且还通过将 CRISPR/Cas9 介导的特异性识别与 RCA 系统的特定 T4 连接反应相结合，协同增强了对 KRAS-G12D 突变的特异性。此外，该系统已成功用于血清中基因组的可视化检测，表明其在临床即时诊断方面具有巨大潜力。
