Automated, portable, and high-throughput fluorescence analyzer (APHF-analyzer) and lateral flow strip based on CRISPR/Cas13a for sensitive and visual detection of SARS-CoV-2

Gaihua Cao; Danqun Huo; Xiaolong Chen; Xianfeng Wang; Shiying Zhou; Shixian Zhao; Xiaogang Luo; Changjun Hou

doi:10.1016/j.talanta.2022.123594

Automated, portable, and high-throughput fluorescence analyzer (APHF-analyzer) and lateral flow strip based on CRISPR/Cas13a for sensitive and visual detection of SARS-CoV-2

Talanta. 2022 Oct 1:248:123594. doi: 10.1016/j.talanta.2022.123594. Epub 2022 May 27.

Authors

Gaihua Cao¹, Danqun Huo², Xiaolong Chen¹, Xianfeng Wang¹, Shiying Zhou¹, Shixian Zhao³, Xiaogang Luo⁴, Changjun Hou⁵

Affiliations

¹ Key Laboratory of Biorheological Science and Technology of Ministry of Education, State and Local Joint Engineering Laboratory for Vascular Implants, Bioengineering College of Chongqing University, Chongqing, 400044, PR China.
² Key Laboratory of Biorheological Science and Technology of Ministry of Education, State and Local Joint Engineering Laboratory for Vascular Implants, Bioengineering College of Chongqing University, Chongqing, 400044, PR China; Chongqing Key Laboratory of Bio-perception & Intelligent Information Processing, School of Microelectronics and Communication Engineering, Chongqing University, Chongqing, 400044, PR China.
³ Chongqing Key Laboratory of Bio-perception & Intelligent Information Processing, School of Microelectronics and Communication Engineering, Chongqing University, Chongqing, 400044, PR China; Chongqing Engineering and Technology Research Center of Intelligent Rehabilitation and Eldercare, Chongqing City Management College, Chongqing, 401331, China. Electronic address: 20161901031@cqu.edu.cn.
⁴ Key Laboratory of Biorheological Science and Technology of Ministry of Education, State and Local Joint Engineering Laboratory for Vascular Implants, Bioengineering College of Chongqing University, Chongqing, 400044, PR China. Electronic address: 13400818@qq.com.
⁵ Key Laboratory of Biorheological Science and Technology of Ministry of Education, State and Local Joint Engineering Laboratory for Vascular Implants, Bioengineering College of Chongqing University, Chongqing, 400044, PR China. Electronic address: houcj@cqu.edu.cn.

Abstract

COVID-19 has erupted and quickly swept across the globe, causing huge losses to human health and wealth. It is of great value to develop a quick, accurate, visual, and high-throughput detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here, we developed a biosensor based on CRISPR/Cas13a combined with recombinase polymerase amplification (RPA) to detect S and Orf1ab genes of SARS-CoV-2 within 30 min. Most important of all, we developed an automated, portable, and high-throughput fluorescence analyzer (APHF-analyzer) with a 3D-printed microfluidic chip for sensitively detecting SARS-CoV-2, which addressed aerosol contamination issue and provided a more accurate and high-throughput detection during the on-site detection process. The detection limits of S gene and Orf1ab gene were as low as 0.68 fM and 4.16 fM. Furthermore, we used the lateral flow strip to realize visualization and point of care testing (POCT) of SARS-CoV-2. Therefore, profit from the efficient amplification of RPA and the high specificity of CRISPR/Cas13a, APHF-analyzer and the lateral flow strip to simultaneous detection of S gene and Orf1ab gene would be applied as a promising tool in the field of SARS-CoV-2 detection.

Keywords: CRISPR/Cas13a; Lateral flow strip; On-site detection; Point of care testing (POCT); Recombinase polymerase amplification (RPA); SARS-CoV-2.

MeSH terms

COVID-19* / diagnosis
Clustered Regularly Interspaced Short Palindromic Repeats
Humans
Nucleic Acid Amplification Techniques
RNA, Viral / genetics
Recombinases
SARS-CoV-2* / genetics
Sensitivity and Specificity

Substances

RNA, Viral
Recombinases