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

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

doi:10.1016/j.bios.2023.115609

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

Biosens Bioelectron. 2023 Nov 1:239:115609. doi: 10.1016/j.bios.2023.115609. Epub 2023 Aug 18.

Authors

Cailing Lin¹, Feng Chen¹, Dongchao Huang¹, Wenyan Li¹, Changsheng He², Yingjun Tang³, Xueping Li⁴, Can Liu⁵, Liya Han¹, Yunpeng Yang¹, Yongchong Zhu¹, Ruikang Chen¹, Yuanju Shi⁴, Chenglai Xia⁵, Zhibin Yan⁶, Hongli Du¹, Lizhen Huang⁷

Affiliations

¹ School of Biology and Biological Engineering, South China University of Technology, Guangzhou, 510006, China.
² School of Biology and Biological Engineering, South China University of Technology, Guangzhou, 510006, China; Experimental Animal Center, Nanfang Hospital, Southern Medical University, Guangzhou, 510515, China.
³ WENS Foodstuff Group Co., Ltd., Yunfu, 527400, China.
⁴ Guangzhou Yoyoung Bio-tech Company, Guangzhou, 510300, China.
⁵ Affiliated Foshan Maternity and Child Healthcare Hospital, Southern Medical University, Foshan, 528000, China; School of Pharmaceutical Sciences, Southern Medical University, Guangzhou, 515150, China.
⁶ Guangdong Provincial Key Laboratory of Optical Information Materials and Technology & Institute of Electronic Paper Displays, South China Academy of Advanced Optoelectronics, South China Normal University, Guangzhou, 510006, PR China.
⁷ School of Biology and Biological Engineering, South China University of Technology, Guangzhou, 510006, China; Fangrui Institute of Innovative Drugs, South China University of Technology, Guangzhou, 510006, China. Electronic address: huanglzh@scut.edu.cn.

PMID: 37611446
DOI: 10.1016/j.bios.2023.115609

Abstract

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.

Keywords: Automation design; CRISPR/Cas12; One-pot; Portable device; Virus detection.

MeSH terms

Biosensing Techniques*
COVID-19* / diagnosis
CRISPR-Cas Systems
DNA
Humans
RNA Viruses*
SARS-CoV-2

Substances

DNA