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2023 |
Fast and visual detection of nucleic acids using a one-step RPA-CRISPR detection (ORCD) system unrestricted by the PAM |
Kangfeng Lin,Jianguang Guo,Xiangju Guo,Qinghan Li,Xiao Li,Zhen Sun,Zehang Zhao,Jiao Weng,Jinzhun Wu,Rui Zhang,Boan Li |
Analytica Chimica Acta |
36813457 |
10.1016/j.aca.2023.340938 |
Fast and visual detection of nucleic acids using a one-step RPA-CRISPR detection (ORCD) system unrestricted by the PAM
Author(s):
Kangfeng Lin,Jianguang Guo,Xiangju Guo,Qinghan Li,Xiao Li,Zhen Sun,Zehang Zhao,Jiao Weng,Jinzhun Wu,Rui Zhang,Boan Li
Journal:
Analytica Chimica Acta
Year:
2023
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.
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