CDetection.v2: One-pot assay for the detection of SARS-CoV-2

Xinge Wang; Yangcan Chen; Xuejia Cheng; Si-Qi Wang; Yanping Hu; Yingmei Feng; Ronghua Jin; Kangping Zhou; Ti Liu; Jianxing Wang; Kai Pan; Bing Liu; Jie Xiang; Yanping Wang; Qi Zhou; Ying Zhang; Weiye Pan; Wei Li

doi:10.3389/fmicb.2023.1158163

CDetection.v2: One-pot assay for the detection of SARS-CoV-2

Front Microbiol. 2023 Mar 23:14:1158163. doi: 10.3389/fmicb.2023.1158163. eCollection 2023.

Authors

Xinge Wang^{1

2

3

4}, Yangcan Chen^{1

2

3

4}, Xuejia Cheng⁵, Si-Qi Wang^{1

2

3}, Yanping Hu^{1

2

3

4}, Yingmei Feng⁶, Ronghua Jin⁷, Kangping Zhou⁸, Ti Liu⁹, Jianxing Wang⁹, Kai Pan⁸, Bing Liu¹⁰, Jie Xiang¹¹, Yanping Wang¹², Qi Zhou^{1

2

3

4}, Ying Zhang^{1

2

3}, Weiye Pan⁵, Wei Li^{1

2

3

4}

Affiliations

¹ State Key Laboratory of Stem Cell and Reproductive Biology, Chinese Academy of Sciences, Institute of Zoology, Beijing, China.
² Bejing Institute for Stem Cell and Regenerative Medicine, Beijing, China.
³ Chinese Academy of Sciences, Institute for Stem Cell and Regenerative Medicine, Beijing, China.
⁴ Savaid Medical School, University of Chinese Academy of Sciences, Beijing, China.
⁵ Beijing SynsorBio Technology Co., Ltd., Beijing, China.
⁶ Department of Science and Technology, Beijing Youan Hospital, Capital Medical University, Beijing, China.
⁷ Beijing Ditan Hospital, Capital Medical University, Beijing, China.
⁸ Hubei Provincial Center for Disease Control and Prevention, Wuhan, China.
⁹ Shandong Center for Disease Control and Prevention, Jinan, China.
¹⁰ Tonghua Central Hospital, Tonghua, Jilin, China.
¹¹ Tongji Medical College of Huazhang, Wuhan Jinyintan Hospital, University of Science and Technology, Wuhan, China.
¹² Hubei Provincial Hospital of Traditional Chinese Medicine, Wuhan, China.

Abstract

Introduction: The ongoing 2019 coronavirus disease pandemic (COVID-19), caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and its variants, is a global public health threat. Early diagnosis and identification of SARS-CoV-2 and its variants plays a critical role in COVID-19 prevention and control. Currently, the most widely used technique to detect SARS-CoV-2 is quantitative reverse transcription real-time quantitative PCR (RT-qPCR), which takes nearly 1 hour and should be performed by experienced personnel to ensure the accuracy of results. Therefore, the development of a nucleic acid detection kit with higher sensitivity, faster detection and greater accuracy is important.

Methods: Here, we optimized the system components and reaction conditions of our previous detection approach by using RT-RAA and Cas12b.

Results: We developed a Cas12b-assisted one-pot detection platform (CDetection.v2) that allows rapid detection of SARS-CoV-2 in 30 minutes. This platform was able to detect up to 5,000 copies/ml of SARS-CoV-2 without cross-reactivity with other viruses. Moreover, the sensitivity of this CRISPR system was comparable to that of RT-qPCR when tested on 120 clinical samples.

Discussion: The CDetection.v2 provides a novel one-pot detection approach based on the integration of RT-RAA and CRISPR/Cas12b for detecting SARS-CoV-2 and screening of large-scale clinical samples, offering a more efficient strategy for detecting various types of viruses.

Keywords: CRISPR/Cas12b; SARS-CoV-2; clinical samples; molecular diagnosis; one-pot detection platform.

Grants and funding

This work was supported by the National Key Research and Development Program (2020YFA0707900, 2018YFA0108400, and 2019YFA0903800), the National Natural Science Foundation of China (31621004 to QZ and WL), and the CAS Project for Young Scientists in Basic Research (YSBR-012 to WL).