Department of Mechanical Engineering, Rochester Institute of Technology, Rochester, New York 14623, United States.
HJ Science & Technology Inc., Berkeley, California 94710, United States.
ACS Appl Mater Interfaces. 2020 Sep 30;12(39):43435-43443. doi: 10.1021/acsami.0c12482. Epub 2020 Sep 17.
We have developed a novel detection system that couples clustered regularly interspaced short palindromic repeat-Cas recognition of target sequences, Cas-mediated nucleic acid probe cleavage, and quantum dots as highly sensitive reporter molecules for simple detection of viral nucleic acid targets. After target recognition and Cas-mediated cleavage of biotinylated ssDNA probe molecules, the probe molecules are bound to magnetic beads. A complementary ssDNA oligonucleotide quantum dot conjugate is then added, which only hybridizes to uncleaved probes on the magnetic beads. After separating hybridized quantum dots, the collected supernatant is illuminated by a portable ultraviolet flashlight, and it provides a simple "Yes-or-No" nucleic acid detection answer. By using a DNA target matching part of the African swine fever virus, detection limits of ∼0.5 and ∼1.25 nM are achieved in buffer and porcine plasma, respectively. The positive samples are readily confirmed by visual inspection, completely avoiding the need for complicated devices and instruments. This work establishes the feasibility of a simple assay for nucleic acid screening in both hospitals and point-of-care settings.
我们开发了一种新颖的检测系统，该系统将靶向序列的簇状规律间隔短回文重复（CRISPR）-Cas 识别、Cas 介导的核酸探针切割与量子点结合在一起，作为用于简单检测病毒核酸靶标的高灵敏度报告分子。在靶标识别和 Cas 介导的生物素化 ssDNA 探针分子切割后，探针分子与磁性珠结合。然后添加互补的 ssDNA 寡核苷酸量子点缀合物，该缀合物仅与磁性珠上未切割的探针杂交。杂交量子点分离后，收集的上清液用便携式紫外手电筒照射，提供简单的“是或否”核酸检测答案。通过使用与非洲猪瘟病毒部分匹配的 DNA 靶标，在缓冲液和猪血浆中分别实现了约 0.5 和 1.25 nM 的检测限。阳性样本通过目视检查即可轻松确认，完全避免了对复杂设备和仪器的需求。这项工作为医院和即时护理环境中的核酸筛选建立了简单检测方法的可行性。
