A universal all-in-one RPA-Cas12a strategy with de novo autodesigner and its application in on-site ultrasensitive detection of DNA and RNA viruses.

Revolutionary all-in-one RPA-CRISPR assays are rapidly becoming the most sought-after tools for point-of-care testing (POCT) due to their high sensitivity and ease of use. Despite the availability of one-pot methods for specific targets, the development of more efficient methods for new targets remains a significant challenge. In this study, we present a rapid and universal approach to establishing an all-in-one RPA-Cas12a method CORDSv2 based on rational balancing amplification and Cas12a cleavage, which achieves ultrasensitive detection of several targets, including SARS-CoV-2, ASFV, HPV16, and HPV18. CORDSv2 demonstrates a limit of detection (LOD) of 0.6 cp/μL and 100% sensitivity for SARS-CoV-2, comparable to qPCR. Combining with our portable device(hippo-CORDS), it has a visual detection LOD of 6 cp/μL and a sensitivity up to 100% for SARS-CoV-2 and 97% for Ct<35 ASFV samples, surpassing most one-pot visual methods. To simplify and accelerate the process for new targets, we also develop a de novo autodesigner by which the optimal couples of primers and crRNA can be selected rapidly. As a universal all-in-one RPA-CRISPR method for on-site testing, CORDSv2 becomes an attractive choice for rapid and accurate diagnosis in resource-limited settings.