Rapid detection of SARS-CoV-2 RNA in saliva via Cas13,Nature Biomedical Engineering

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Rapid detection of SARS-CoV-2 RNA in saliva via Cas13
Nature Biomedical Engineering ( IF 26.8 ) Pub Date : 2022-08-11 , DOI: 10.1038/s41551-022-00917-y
Sita S Chandrasekaran _{1,

2,

3} , Shreeya Agrawal _{1,

2} , Alison Fanton _{1,

2,

3} , Aditya R Jangid _{1,

2} , Bérénice Charrez ₃ , Arturo M Escajeda ₄ , Sungmin Son ₁ , Roger Mcintosh ₄ , Huyen Tran ₄ , Abdul Bhuiya _{1,

3} , María Díaz de León Derby _{1,

3} , Neil A Switz ₅ , Maxim Armstrong ₆ , Andrew R Harris ₁ , Noam Prywes ₂ , Maria Lukarska _{2,

7} , Scott B Biering ₈ , Dylan C J Smock _{2,

7} , Amanda Mok ₉ , Gavin J Knott _{2,

7,

10} , Qi Dang ₂ , Erik Van Dis ₇ , Eli Dugan _{2,

7} , Shin Kim _{2,

7} , Tina Y Liu _{2,

7} , , Erica A Moehle ₂ , Katherine Kogut ₁₁ , Brenda Eskenazi ₁₁ , Eva Harris ₈ , Sarah A Stanley _{7,

12} , Liana F Lareau _{1,

2} , Ming X Tan ₄ , Daniel A Fletcher ₁ , Jennifer A Doudna _{2,

6,

7,

13,

14,

15} , David F Savage ₇ , Patrick D Hsu _{1,

2,

16}

Affiliation

Department of Bioengineering, University of California, Berkeley, Berkeley, CA, USA.
Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA, USA.
University of California, Berkeley-University of California, San Francisco Graduate Program in Bioengineering, Berkeley, CA, USA.
Wainamics Inc., Pleasanton, CA, USA.
Department of Physics and Astronomy, San José State University, San José, CA, USA.
Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA.
Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA, USA.
Division of Infectious Diseases and Vaccinology, School of Public Health, University of California, Berkeley, Berkeley, CA, USA.
Center for Computational Biology, University of California, Berkeley, Berkeley, CA, USA.
Monash Biomedicine Discovery Institute, Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia.
Center for Environmental Research and Community Health (CERCH), School of Public Health, University of California, Berkeley, Berkeley, CA, USA.
School of Public Health, University of California, Berkeley, Berkeley, CA, USA.
Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, CA, USA.
Department of Chemistry, University of California, Berkeley, Berkeley, CA, USA.
Gladstone Institute of Data Science and Biotechnology, Gladstone Institutes, San Francisco, CA, USA.
Arc Institute, Palo Alto, CA, USA.

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^–1, 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.

中文翻译：

通过 Cas13 快速检测唾液中的 SARS-CoV-2 RNA

快速核酸检测是传染病监测的核心。在这里，我们报告了一种快速检测 COVID-19 的方法及其在原型微流体设备中的实施。该检测方法被我们命名为 DISCoVER（用于冠状病毒酶报告诊断），包括通过货架稳定且低成本的试剂进行免提取样品裂解、多重等温 RNA 扩增、随后的 T7 转录以及 Cas13 介导的猝灭荧光团裂解。该设备由插入紧凑型仪器的一次性重力驱动微流体盒组成，可在 60 分钟内自动运行测定并读出荧光。 DISCoVER 可以检测唾液中的严重急性呼吸综合征冠状病毒 2 (SARS-CoV-2)，灵敏度为 40 个拷贝 μl ^–1 ，使用从 63 个样本中提取的总 RNA 进行验证（针对定量 PCR）时，灵敏度为 94%，特异性为 100%鼻拭子样本（33 个 SARS-CoV-2 阳性，周期阈值为 13-35）。该设备正确识别了所有测试的临床唾液样本（13 个样本中有 10 个样本呈 SARS-CoV-2 阳性，循环阈值为 23-31）。快速护理点核酸检测可能会扩大分子诊断的用途。

更新日期：2022-08-11

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