An engineered CRISPR-Cas12a variant and DNA-RNA hybrid guides enable robust and rapid COVID-19 testing.
Nat Commun
; 12(1): 1739, 2021 03 19.
Article
in En
| MEDLINE
| ID: mdl-33741959
ABSTRACT
Extensive testing is essential to break the transmission of SARS-CoV-2, which causes the ongoing COVID-19 pandemic. Here, we present a CRISPR-based diagnostic assay that is robust to viral genome mutations and temperature, produces results fast, can be applied directly on nasopharyngeal (NP) specimens without RNA purification, and incorporates a human internal control within the same reaction. Specifically, we show that the use of an engineered AsCas12a enzyme enables detection of wildtype and mutated SARS-CoV-2 and allows us to perform the detection step with loop-mediated isothermal amplification (LAMP) at 60-65 °C. We also find that the use of hybrid DNA-RNA guides increases the rate of reaction, enabling our test to be completed within 30 minutes. Utilizing clinical samples from 72 patients with COVID-19 infection and 57 healthy individuals, we demonstrate that our test exhibits a specificity and positive predictive value of 100% with a sensitivity of 50 and 1000 copies per reaction (or 2 and 40 copies per microliter) for purified RNA samples and unpurified NP specimens respectively.
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
RNA, Guide, Kinetoplastida
/
COVID-19 Testing
/
SARS-CoV-2
/
COVID-19
Type of study:
Diagnostic_studies
/
Prognostic_studies
Limits:
Humans
Language:
En
Journal:
Nat Commun
Journal subject:
BIOLOGIA
/
CIENCIA
Year:
2021
Document type:
Article
Affiliation country:
Singapur