Fast and visual detection of nucleic acids using a one-step RPA-CRISPR detection (ORCD) system unrestricted by the PAM.
Anal Chim Acta
; 1248: 340938, 2023 Apr 01.
Article
in English
| MEDLINE | ID: covidwho-2242732
ABSTRACT
CRISPR-Cas12a (Cpf1) is widely used for pathogen detection. However, most Cas12a nucleic acid detection methods are limited by a PAM sequence requirement. Moreover, preamplification and Cas12a cleavage are separate. Here, we developed a one-step RPA-CRISPR detection (ORCD) system unrestricted by the PAM sequence with high sensitivity and specificity that offers one-tube, rapid, and visually observable detection of nucleic acids. In this system, Cas12a detection and RPA amplification are performed simultaneously, without separate preamplification and product transfer steps, and 0.2 copies/µL of DNA and 0.4 copies/µL of RNA can be detected. In the ORCD system, the activity of Cas12a is the key to the nucleic acid detection; specifically, reducing Cas12a activity increases the sensitivity of ORCD assay detection of the PAM target. Furthermore, by combining this detection technique with a nucleic acid extraction-free method, our ORCD system can be used to extract, amplify and detect samples within 30 min, as verified with tests of 82 Bordetella pertussis clinical samples with a sensitivity and specificity of 97.30% and 100% compared with PCR. We also tested 13 SARS-CoV-2 samples with RT-ORCD, and the results were consistent with RT-PCR.
Keywords
Full text:
Available
Collection:
International databases
Database:
MEDLINE
Main subject:
Nucleic Acids
/
COVID-19
Type of study:
Diagnostic study
/
Prognostic study
Limits:
Humans
Language:
English
Journal:
Anal Chim Acta
Year:
2023
Document Type:
Article
Affiliation country:
J.aca.2023.340938
Similar
MEDLINE
...
LILACS
LIS