Fast and visual detection of nucleic acids using a one-step RPA-CRISPR detection (ORCD) system unrestricted by the PAM

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/mu L of DNA and 0.4 copies/mu L 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.

Automated summary

We developed a one-step RPA-CRISPR detection system (ORCD) that can detect nucleic acids with high sensitivity and specificity without restriction of the PAM sequence. In this system, Cas12a detection and RPA amplification are performed simultaneously, and 0.2 copies/μL of DNA and 0.4 copies/μL of RNA can be detected. By combining this detection technique with a nucleic acid extraction-free method, our ORCD system can extract, amplify, and detect samples within 30 minutes, and shows high sensitivity and specificity compared to PCR.