School of Biomolecular Science and Engineering, Vidyasirimedhi Institute of Science and Technology (VISTEC).
School of Biomolecular Science and Engineering, Vidyasirimedhi Institute of Science and Technology (VISTEC);
J Vis Exp. 2024 Aug 16(210). doi: 10.3791/66703.
Molecular diagnostics by Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based detection have high diagnostic accuracy and attributes that are suitable for use at point-of-care settings such as fast turnaround times for results, convenient simple readouts, and no requirement of complicated instruments. However, the reactions can be cumbersome to perform at the point of care due to their many components and manual handling steps. Herein, we provide a step-by-step, optimized protocol for the robust detection of disease pathogens and genetic markers with recombinase-based isothermal amplification and CRISPR-based reagents, which are premixed and then freeze-dried in easily stored and ready-to-use formats. Premixed, freeze-dried reagents can be rehydrated for immediate use and retain high amplification and detection efficiencies. We also provide a troubleshooting guide for commonly found problems upon preparing and using premixed, freeze-dried reagents for CRISPR-based diagnostics, to make the detection platform more accessible to the wider diagnostic/genetic testing communities.
基于簇状规律间隔短回文重复序列（CRISPR）的分子诊断具有很高的诊断准确性，并且具有适合在即时检测（point-of-care）环境下使用的特点，例如结果的快速周转时间、方便简单的读数以及不需要复杂的仪器。然而，由于其许多组件和手动操作步骤，反应在即时检测时可能很繁琐。在此，我们提供了一个基于重组酶的等温扩增和基于 CRISPR 的试剂的稳健检测疾病病原体和遗传标记的分步、优化方案，这些试剂预先混合，然后以易于储存和即用型格式冻干。预混合、冻干的试剂可以再水化以供立即使用，并保持高的扩增和检测效率。我们还为在基于 CRISPR 的诊断中制备和使用预混合、冻干试剂时常见的问题提供了故障排除指南，以使检测平台更容易被更广泛的诊断/遗传测试社区所接受。
