Department of Biomedical Engineering, University of Connecticut Health Center, 263 Farmington Ave., Farmington, CT, 06030, USA.
Section of Oral Medicine, University of Connecticut Health Center, Farmington, CT, 06030, USA.
Nat Commun. 2020 Sep 18;11(1):4711. doi: 10.1038/s41467-020-18575-6.
The recent outbreak of novel coronavirus (SARS-CoV-2) causing COVID-19 disease spreads rapidly in the world. Rapid and early detection of SARS-CoV-2 facilitates early intervention and prevents the disease spread. Here, we present an All-In-One Dual CRISPR-Cas12a (AIOD-CRISPR) assay for one-pot, ultrasensitive, and visual SARS-CoV-2 detection. By targeting SARS-CoV-2's nucleoprotein gene, two CRISPR RNAs without protospacer adjacent motif (PAM) site limitation are introduced to develop the AIOD-CRISPR assay and detect the nucleic acids with a sensitivity of few copies. We validate the assay by using COVID-19 clinical swab samples and obtain consistent results with RT-PCR assay. Furthermore, a low-cost hand warmer (~$0.3) is used as an incubator of the AIOD-CRISPR assay to detect clinical samples within 20 min, enabling an instrument-free, visual SARS-CoV-2 detection at the point of care. Thus, our method has the significant potential to provide a rapid, sensitive, one-pot point-of-care assay for SARS-CoV-2.
新型冠状病毒（SARS-CoV-2）引发的 COVID-19 疾病在全球迅速传播。快速和早期检测 SARS-CoV-2 有助于早期干预和防止疾病传播。在这里，我们提出了一种用于一锅、超灵敏和可视化 SARS-CoV-2 检测的一体式双重 CRISPR-Cas12a（AIOD-CRISPR）检测方法。通过靶向 SARS-CoV-2 的核蛋白基因，引入了两个没有前导序列相邻基序（PAM）位点限制的 CRISPR RNA，以开发 AIOD-CRISPR 检测方法并检测具有低拷贝数灵敏度的核酸。我们使用 COVID-19 临床拭子样本验证了该检测方法，并与 RT-PCR 检测方法获得了一致的结果。此外，低成本的暖手器（约 0.3 美元）被用作 AIOD-CRISPR 检测方法的孵化器，可在 20 分钟内检测临床样本，实现了无仪器、在护理点进行可视化 SARS-CoV-2 检测。因此，我们的方法具有提供快速、敏感、一锅式即时护理点 SARS-CoV-2 检测的巨大潜力。
