Department of Biomedical Engineering, School of Medicine, Tsinghua University, Beijing 100084, China.
Department of Biochemistry and Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and School of Basic Medicine, Peking Union Medical College, Beijing 100730, China.
ACS Sens. 2021 Nov 26;6(11):4057-4066. doi: 10.1021/acssensors.1c01491. Epub 2021 Oct 25.
Nucleic acid detection plays a vital role in both biomedical research and clinical medicine. The temperature circulation changes of the widely used polymerase chain reaction technique are time-consuming and technically challenging for system development. Recombinase polymerase amplification (RPA) is an isothermal method for rapid nucleic acid detection. However, current RPA amplicon detection methods are complicated and expensive and easily generate false positives, restricting the promotion of RPA techniques. In this work, a hyperspectral interferometric amplicon-complex quantitation method is presented, combined with asymmetric dipole complex strategy optical scattering analysis. GelRed dye was utilized to form amplicon-complex particles, and the Fourier domain spectrum computation contributed to complex scattering quantitation. With this method, a supporting microfluidic chip and automatic system were developed to achieve integrated, rapid, quantitative, and miniscule nucleic acid detection. The gene was utilized as an example for targeted nucleic acid quantitation and single nucleotide polymorphism detection. The total reaction time was decreased to merely 20 min, and the limit of detection was only 3.17 ng/μL. The minimum measurable concentration of target was 1.68 copies/μL, 31.67 times more sensitive than turbidity detection, and the single reaction chamber was only 9.33 μL. No scattering increase occurred for template-free control, and thus, false positives caused by primer dimers and nonspecific products could be avoided. The experimental results prove that the provided method and system can detect single-base mutations in the gene and is a reasonable technique for rapid, automatic, and low-cost nucleic acid detection.
核酸检测在生物医学研究和临床医学中都起着至关重要的作用。广泛应用的聚合酶链反应技术的温度循环变化既耗时又对系统开发具有技术挑战性。重组酶聚合酶扩增（RPA）是一种用于快速核酸检测的等温方法。然而，目前的 RPA 扩增子检测方法复杂且昂贵，容易产生假阳性，限制了 RPA 技术的推广。在这项工作中，提出了一种超光谱干涉扩增子复合定量方法，结合不对称偶极子复合物策略的光散射分析。利用 GelRed 染料形成扩增子复合物颗粒，傅里叶域光谱计算有助于复杂散射定量。利用这种方法，开发了一个支持微流控芯片和自动系统，实现了集成、快速、定量和微小的核酸检测。以基因为例进行了靶向核酸定量和单核苷酸多态性检测。总反应时间缩短至仅 20 分钟，检测限仅为 3.17ng/μL。目标的最小可测量浓度为 1.68 拷贝/μL，比浊度检测灵敏 31.67 倍，单个反应室仅为 9.33μL。无模板对照没有散射增加，因此可以避免引物二聚体和非特异性产物引起的假阳性。实验结果证明，所提供的方法和系统可以检测基因中的单碱基突变，是一种快速、自动、低成本的核酸检测的合理技术。
