Laboratory for Genome Engineering and Synthetic Biology, Division of Biological Sciences, King Abdullah University of Science and Technology (KAUST), Thuwal 23955-6900, Saudi Arabia.
Laboratory of DNA Replication and Recombination, Biological and Environmental Sciences and Engineering Division, King Abdullah University of Science and Technology (KAUST), Thuwal 23955-6900, Saudi Arabia.
Nano Lett. 2021 Apr 28;21(8):3596-3603. doi: 10.1021/acs.nanolett.1c00612. Epub 2021 Apr 12.
Rapid, sensitive, and specific point-of-care testing for pathogens is crucial for disease control. Lateral flow assays (LFAs) have been employed for nucleic acid detection, but they have limited sensitivity and specificity. Here, we used a fusion of catalytically inactive SpCas9 endonuclease and VirD2 relaxase for sensitive, specific nucleic acid detection by LFA. In this assay, the target nucleic acid is amplified with biotinylated oligos. VirD2-dCas9 specifically binds the target sequence via dCas9 and covalently binds to a FAM-tagged oligonucleotide via VirD2. The biotin label and FAM tag are detected by a commercially available LFA. We coupled this system, named Vigilant (rD2-dCas9 uded and FA-coupled ucleic acid est), to reverse transcription-recombinase polymerase amplification to detect SARS-CoV2 in clinical samples. Vigilant exhibited a limit of detection of 2.5 copies/μL, comparable to CRISPR-based systems, and showed no cross-reactivity with SARS-CoV1 or MERS. Vigilant offers an easy-to-use, rapid, cost-effective, and robust detection platform for SARS-CoV2.
快速、灵敏、特异的即时检测对于疾病控制至关重要。侧向流检测（LFA）已被用于核酸检测，但它们的灵敏度和特异性有限。在这里，我们使用无酶活性的 SpCas9 内切酶和 VirD2 解旋酶的融合来通过 LFA 进行灵敏、特异的核酸检测。在该检测中，目标核酸通过生物素化寡核苷酸进行扩增。VirD2-dCas9 通过 dCas9 特异性结合靶序列，并通过 VirD2 共价结合到 FAM 标记的寡核苷酸上。生物素标记和 FAM 标记通过市售的 LFA 进行检测。我们将该系统命名为 Vigilant（rD2-dCas9 与 FA 偶联的核酸 est），并与逆转录-重组酶聚合酶扩增（RT-RPA）结合，以检测临床样本中的 SARS-CoV2。Vigilant 的检测限为 2.5 拷贝/μL，与基于 CRISPR 的系统相当，且与 SARS-CoV1 或 MERS 无交叉反应。Vigilant 为 SARS-CoV2 提供了一种易于使用、快速、具有成本效益且强大的检测平台。
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