Zhang Jiaxing, Hui Hui, Xu Wei, Hua Kai, Cui Yali, Liu Xiaonan
College of Life Sciences, Northwest University Xi'an 710069 China
Department of Immunology, The Fourth Military Medical University Xi'an 710032 China.
RSC Adv. 2022 Aug 10;12(34):22091-22096. doi: 10.1039/d2ra03989c. eCollection 2022 Aug 4.
Genetic variation plays a crucial role in disease occurrence and development. However, current genotyping strategies not only require a long turnaround time for DNA purification, but also depend on sophisticated apparatus and complex data interpretation, which seriously limits their application in point of care diagnostic test scenarios. In this study, by integrating phosphate induced coloration reaction and loop-mediated isothermal amplification, a rapid and portable strategy for straightforward genotyping has been established to cater to the demand of precision medicine. By employing phosphate ions produced during the amplification as a signal generator, not only can the genotyping result be interpreted with only naked eye from a low-cost label-free strip, but also the amplification efficiency is increased to facilitate genotyping with a robust biological specimen ignoring DNA polymerase inhibitors. Moreover, the introduction of alkaline lysis for DNA release allows whole blood to be identified accurately avoiding DNA purification. As a proof of concept, the insertion/deletion polymorphisms of the angiotensin-converting enzyme, a crucial factor associated with cardiovascular and cerebrovascular diseases, has been selected as a model to evaluate the performance of this method. Accurate results can be obtained from as low as 1 ng genomic DNA within 30 min. For clinical specimen detection, a concordance rate up to 100% has been found compared with PCR-based electrophoresis. Thus, this novel strategy may serve as a promising tool for straightforward genotyping to provide timely diagnostic information, especially in resource-poor medical institutions.
基因变异在疾病的发生和发展中起着至关重要的作用。然而，当前的基因分型策略不仅需要较长的DNA纯化周转时间，而且依赖于精密的仪器和复杂的数据解读，这严重限制了它们在即时诊断测试场景中的应用。在本研究中，通过整合磷酸盐诱导显色反应和环介导等温扩增技术，建立了一种快速且便携的直接基因分型策略，以满足精准医学的需求。通过将扩增过程中产生的磷酸根离子用作信号发生器，不仅可以通过低成本的无标记试纸条仅用肉眼解读基因分型结果，而且还提高了扩增效率，以便在存在DNA聚合酶抑制剂的情况下仍能使用生物样本进行稳健的基因分型。此外，引入用于释放DNA的碱性裂解方法可避免DNA纯化，从而准确鉴定全血。作为概念验证，选择与心脑血管疾病相关的关键因素血管紧张素转换酶的插入/缺失多态性作为模型来评估该方法的性能。在30分钟内，低至1 ng的基因组DNA即可获得准确结果。对于临床样本检测，与基于PCR的电泳相比，一致性率高达100%。因此，这种新策略可能成为直接基因分型的有前途的工具，以提供及时的诊断信息，特别是在资源匮乏的医疗机构中。
