通过 Cas13 快速检测唾液中的 SARS-CoV-2 RNA。
Rapid detection of SARS-CoV-2 RNA in saliva via Cas13.
机构信息
Department of Bioengineering, University of California, Berkeley, Berkeley, CA, USA.
Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA, USA.
出版信息
Nat Biomed Eng. 2022 Aug;6(8):944-956. doi: 10.1038/s41551-022-00917-y. Epub 2022 Aug 11.
Rapid nucleic acid testing is central to infectious disease surveillance. Here, we report an assay for rapid COVID-19 testing and its implementation in a prototype microfluidic device. The assay, which we named DISCoVER (for diagnostics with coronavirus enzymatic reporting), involves extraction-free sample lysis via shelf-stable and low-cost reagents, multiplexed isothermal RNA amplification followed by T7 transcription, and Cas13-mediated cleavage of a quenched fluorophore. The device consists of a single-use gravity-driven microfluidic cartridge inserted into a compact instrument for automated running of the assay and readout of fluorescence within 60 min. DISCoVER can detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in saliva with a sensitivity of 40 copies μl, and was 94% sensitive and 100% specific when validated (against quantitative PCR) using total RNA extracted from 63 nasal-swab samples (33 SARS-CoV-2-positive, with cycle-threshold values of 13-35). The device correctly identified all tested clinical saliva samples (10 SARS-CoV-2-positive out of 13, with cycle-threshold values of 23-31). Rapid point-of-care nucleic acid testing may broaden the use of molecular diagnostics.
快速核酸检测是传染病监测的核心。在这里，我们报告了一种用于快速 COVID-19 检测的方法及其在原型微流控设备中的实施。该检测方法被我们命名为 DISCoVER（用于冠状病毒酶报告的诊断），它涉及通过稳定货架且低成本的试剂进行无提取样本裂解，然后进行多重等温 RNA 扩增，接着进行 T7 转录，以及 Cas13 介导的淬灭荧光团的切割。该设备由一个一次性重力驱动微流控盒组成，插入到一个紧凑的仪器中，用于自动运行检测，并在 60 分钟内读取荧光。DISCoVER 可以用灵敏度为 40 拷贝 μl 的唾液检测到严重急性呼吸综合征冠状病毒 2（SARS-CoV-2），并在使用从 63 个鼻拭子样本（33 个 SARS-CoV-2 阳性，循环阈值为 13-35）提取的总 RNA 进行验证（与定量 PCR 相比）时，其敏感性为 94%，特异性为 100%。该设备正确识别了所有测试的临床唾液样本（13 个中有 10 个 SARS-CoV-2 阳性，循环阈值为 23-31）。快速即时核酸检测可能会扩大分子诊断的应用范围。
Zotero 插件