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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  

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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