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

Authors：Lin, Cailing; Chen, Feng; Huang, Dongchao^*; Li, Wenyan; He, Changsheng; Tang, Yingjun; Li, Xueping; Liu, Can; Han, Liya; Yang, Yunpeng; Zhu, Yongchong; Chen, Ruikang; Shi, Yuanju; Xia, Chenglai; Yan, Zhibin; Du, Hongli; Huang, Lizhen^*

Source：Biosensors and Bioelectronics, 2023, 239: 115609.

DOI：10.1016/j.bios.2023.115609

• One-pot
• CRISPR/Cas12
• Virus detection
• Portable device
• Automation design

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/mu L 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/mu L 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.

• Institution
  南方医科大学