EBioMedicine (Nov 2020)
A CRISPR-Cas12a-based specific enhancer for more sensitive detection of SARS-CoV-2 infection
- Weiren Huang,
- Lei Yu,
- Donghua Wen,
- Dong Wei,
- Yangyang Sun,
- Huailong Zhao,
- Yu Ye,
- Wei Chen,
- Yongqiang Zhu,
- Lijun Wang,
- Li Wang,
- Wenjuan Wu,
- Qianqian Zhao,
- Yong Xu,
- Dayong Gu,
- Guohui Nie,
- Dongyi Zhu,
- Zhongliang Guo,
- Xiaoling Ma,
- Liman Niu,
- Yikun Huang,
- Yuchen Liu,
- Bo Peng,
- Renli Zhang,
- Xiuming Zhang,
- Dechang Li,
- Yang Liu,
- Guoliang Yang,
- Lanzheng Liu,
- Yunying Zhou,
- Yunshan Wang,
- Tieying Hou,
- Qiuping Gao,
- Wujiao Li,
- Shuo Chen,
- Xuejiao Hu,
- Mei Han,
- Huajun Zheng,
- Jianping Weng,
- Zhiming Cai,
- Xinxin Zhang,
- Fei Song,
- Guoping Zhao,
- Jin Wang
Affiliations
- Weiren Huang
- Department of Urology, Shenzhen Second People's Hospital, The First Affiliated Hospital of Shenzhen University, International Cancer Center, Shenzhen University School of Medicine, Shenzhen 518039, China
- Lei Yu
- Department of Urology, Shenzhen Second People's Hospital, The First Affiliated Hospital of Shenzhen University, International Cancer Center, Shenzhen University School of Medicine, Shenzhen 518039, China
- Donghua Wen
- Department of Laboratory Medicine, Shanghai East Hospital, Tongji University School of Medicine, Shanghai 200123, China
- Dong Wei
- Research Laboratory of Clinical Virology, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai 200025, China
- Yangyang Sun
- Department of Urology, Shenzhen Second People's Hospital, The First Affiliated Hospital of Shenzhen University, International Cancer Center, Shenzhen University School of Medicine, Shenzhen 518039, China
- Huailong Zhao
- Jinan Center for Disease Control and Prevention, Jinan, Shandong 250021, China
- Yu Ye
- Shenzhen Institute of Synthetic Biology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, China
- Wei Chen
- Department of Urology, Shenzhen Second People's Hospital, The First Affiliated Hospital of Shenzhen University, International Cancer Center, Shenzhen University School of Medicine, Shenzhen 518039, China
- Yongqiang Zhu
- Shanghai-MOST Key Laboratory of Health and Disease Genomics, Chinese National Human Genome Center at Shanghai, Shanghai, 201203, China
- Lijun Wang
- Shanghai-MOST Key Laboratory of Health and Disease Genomics, Chinese National Human Genome Center at Shanghai, Shanghai, 201203, China
- Li Wang
- Jinan Infectious Diseases Hospital of Shandong University, Jinan, Shandong 250021, China
- Wenjuan Wu
- Department of Laboratory Medicine, Shanghai East Hospital, Tongji University School of Medicine, Shanghai 200123, China
- Qianqian Zhao
- Medical Research & Laboratory Diagnostic Center, Jinan Central Hospital Affiliated to Shandong University, Jinan, Shandong 250013, China
- Yong Xu
- Department of Urology, Shenzhen Second People's Hospital, The First Affiliated Hospital of Shenzhen University, International Cancer Center, Shenzhen University School of Medicine, Shenzhen 518039, China
- Dayong Gu
- Department of Urology, Shenzhen Second People's Hospital, The First Affiliated Hospital of Shenzhen University, International Cancer Center, Shenzhen University School of Medicine, Shenzhen 518039, China
- Guohui Nie
- Department of Urology, Shenzhen Second People's Hospital, The First Affiliated Hospital of Shenzhen University, International Cancer Center, Shenzhen University School of Medicine, Shenzhen 518039, China
- Dongyi Zhu
- Department of Laboratory Medicine, Shanghai East Hospital, Tongji University School of Medicine, Shanghai 200123, China
- Zhongliang Guo
- Department of Laboratory Medicine, Shanghai East Hospital, Tongji University School of Medicine, Shanghai 200123, China
- Xiaoling Ma
- The First Affiliated Hospital of USTC, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, Anhui 230001, China
- Liman Niu
- Department of Urology, Shenzhen Second People's Hospital, The First Affiliated Hospital of Shenzhen University, International Cancer Center, Shenzhen University School of Medicine, Shenzhen 518039, China
- Yikun Huang
- Department of Urology, Shenzhen Second People's Hospital, The First Affiliated Hospital of Shenzhen University, International Cancer Center, Shenzhen University School of Medicine, Shenzhen 518039, China
- Yuchen Liu
- Department of Urology, Shenzhen Second People's Hospital, The First Affiliated Hospital of Shenzhen University, International Cancer Center, Shenzhen University School of Medicine, Shenzhen 518039, China
- Bo Peng
- Shenzhen Center for Disease Control and Prevention, Shenzhen 518055, China
- Renli Zhang
- Shenzhen Center for Disease Control and Prevention, Shenzhen 518055, China
- Xiuming Zhang
- Shenzhen Sixth People's Hospital (Nanshan Hospital), Huazhong University of Science and Technology Union Shenzhen Hospital, Shenzhen 518052, China
- Dechang Li
- Yuebei Second People's Hospital, Shaoguan, Guangdong 512000, China
- Yang Liu
- Shanghai Institute of Quality Inspection and Technical Research/National Quality Supervision and Inspection Center for Food Products (Shanghai), Shanghai 200233, China
- Guoliang Yang
- Jinan Center for Disease Control and Prevention, Jinan, Shandong 250021, China
- Lanzheng Liu
- Jinan Center for Disease Control and Prevention, Jinan, Shandong 250021, China
- Yunying Zhou
- Medical Research & Laboratory Diagnostic Center, Jinan Central Hospital Affiliated to Shandong University, Jinan, Shandong 250013, China
- Yunshan Wang
- Medical Research & Laboratory Diagnostic Center, Jinan Central Hospital Affiliated to Shandong University, Jinan, Shandong 250013, China
- Tieying Hou
- Laboratory of Medicine, Provincial People's Hospital, Guangdong Academy of Medical Sciences Guangzhou, Guangdong 510080, China
- Qiuping Gao
- Tolo Biotechnology Company Limited, Shanghai 200233, China
- Wujiao Li
- Department of Urology, Shenzhen Second People's Hospital, The First Affiliated Hospital of Shenzhen University, International Cancer Center, Shenzhen University School of Medicine, Shenzhen 518039, China
- Shuo Chen
- Shenzhen Institute of Synthetic Biology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, China
- Xuejiao Hu
- Laboratory of Medicine, Provincial People's Hospital, Guangdong Academy of Medical Sciences Guangzhou, Guangdong 510080, China
- Mei Han
- Public Health Medical Centre of Chongqing Municipality, Chongqing 400036, China
- Huajun Zheng
- Shanghai-MOST Key Laboratory of Health and Disease Genomics, Chinese National Human Genome Center at Shanghai, Shanghai, 201203, China
- Jianping Weng
- The First Affiliated Hospital of USTC, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, Anhui 230001, China
- Zhiming Cai
- Department of Urology, Shenzhen Second People's Hospital, The First Affiliated Hospital of Shenzhen University, International Cancer Center, Shenzhen University School of Medicine, Shenzhen 518039, China
- Xinxin Zhang
- Research Laboratory of Clinical Virology, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai 200025, China
- Fei Song
- Department of Urology, Shenzhen Second People's Hospital, The First Affiliated Hospital of Shenzhen University, International Cancer Center, Shenzhen University School of Medicine, Shenzhen 518039, China; Corresponding authors: Department of Urology, Shenzhen Second People’s Hospital, The First Affiliated Hospital of Shenzhen University, International Cancer Center, Shenzhen University School of Medicine, Shenzhen 518039, China.
- Guoping Zhao
- Tolo Biotechnology Company Limited, Shanghai 200233, China; CAS Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China; Bio-Med Big Data Center, Key Laboratory of Computational Biology, CAS-MPG Partner Institute for Computational Biology, Shanghai Institute of Nutrition and Health, Chinese Academy of Sciences, Shanghai 200031, China; Corresponding author: CAS Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China.
- Jin Wang
- College of Life Sciences, Shanghai Normal University, Shanghai 200234, China; Corresponding author: College of Life Sciences, Shanghai Normal University, Shanghai 200234, China.
- Journal volume & issue
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Vol. 61
p. 103036
Abstract
Background: Real-time reverse transcription-PCR (rRT-PCR) has been the most effective and widely implemented diagnostic technology since the beginning of the COVID-19 pandemic. However, fuzzy rRT-PCR readouts with high Ct values are frequently encountered, resulting in uncertainty in diagnosis. Methods: A Specific Enhancer for PCR-amplified Nucleic Acid (SENA) was developed based on the Cas12a trans-cleavage activity, which is specifically triggered by the rRT-PCR amplicons of the SARS-CoV-2 Orf1ab (O) and N fragments. SENA was first characterized to determine its sensitivity and specificity, using a systematic titration experiment with pure SARS-CoV-2 RNA standards, and was then verified in several hospitals, employing a couple of commercial rRT-PCR kits and testing various clinical specimens under different scenarios. Findings: The ratio (10 min/5 min) of fluorescence change (FC) with mixed SENA reaction (mix-FCratio) was defined for quantitative analysis of target O and N genes, and the Limit of Detection (LoD) of mix-FCratio with 95% confidence interval was 1.2≤1.6≤2.1. Totally, 295 clinical specimens were analyzed, among which 21 uncertain rRT-PCR cases as well as 4 false negative and 2 false positive samples were characterized by SENA and further verified by next-generation sequencing (NGS). The cut-off values for mix-FCratio were determined as 1.145 for positive and 1.068 for negative. Interpretation: SENA increases both the sensitivity and the specificity of rRT-PCR, solving the uncertainty problem in COVID-19 diagnosis and thus providing a simple and low-cost companion diagnosis for combating the pandemic. Funding: Detailed funding information is available at the end of the manuscript.
