Rapid detection of SARS-CoV-2 RNA in saliva via Cas13.
Sita S ChandrasekaranShreeya AgrawalAlison FantonAditya R JangidBérénice CharrezArturo M EscajedaSungmin SonRoger McintoshHuyen TranAbdul BhuiyaMaría Díaz de León DerbyNeil A SwitzMaxim ArmstrongAndrew R HarrisNoam PrywesMaria LukarskaScott B BieringDylan C J SmockAmanda MokGavin J KnottQi DangErik Van DisEli DuganShin KimTina Y Liunull nullErica A MoehleKatherine KogutBrenda EskenaziEva HarrisSarah A StanleyLiana F LareauMing X TanDaniel A FletcherJennifer A DoudnaDavid F SavagePatrick D HsuPublished in: Nature biomedical engineering (2022)
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.
Keyphrases
- nucleic acid
- sars cov
- respiratory syndrome coronavirus
- high throughput
- low cost
- single cell
- infectious diseases
- crispr cas
- coronavirus disease
- genome editing
- healthcare
- circulating tumor cells
- primary care
- single molecule
- emergency department
- deep learning
- high resolution
- mass spectrometry
- quality improvement
- energy transfer