A CRISPR-Cas12a-based specific enhancer for more sensitive detection of SARS-CoV-2 infection

被引：35

作者：

Huang, Weiren ^{[1
]}

Yu, Lei ^{[1
]}

Wen, Donghua ^{[2
]}

Wei, Dong ^{[3
]}

Sun, Yangyang ^{[1
]}

Zhao, Huailong ^{[4
]}

Ye, Yu ^{[5
]}

Chen, Wei ^{[1
]}

Zhu, Yongqiang ^{[6
]}

Wang, Lijun ^{[6
]}

Wang, Li ^{[7
]}

Wu, Wenjuan ^{[2
]}

Zhao, Qianqian ^{[8
]}

Xu, Yong ^{[1
]}

Gu, Dayong ^{[1
]}

Nie, Guohui ^{[1
]}

Zhu, Dongyi ^{[2
]}

Guo, Zhongliang ^{[2
]}

Ma, Xiaoling ^{[9
]}

Niu, Liman ^{[1
]}

Huang, Yikun ^{[1
]}

Liu, Yuchen ^{[1
]}

Peng, Bo ^{[10
]}

Zhang, Renli ^{[10
]}

Zhang, Xiuming ^{[11
]}

Li, Dechang ^{[12
]}

Liu, Yang ^{[13
]}

Yang, Guoliang ^{[4
]}

Liu, Lanzheng ^{[4
]}

Zhou, Yunying ^{[8
]}

Wang, Yunshan ^{[8
]}

Hou, Tieying ^{[14
]}

Gao, Qiuping ^{[15
]}

Li, Wujiao ^{[1
]}

Chen, Shuo ^{[5
]}

Hu, Xuejiao ^{[14
]}

Han, Mei ^{[16
]}

Zheng, Huajun ^{[6
]}

Weng, Jianping ^{[9
]}

Cai, Zhiming ^{[1
]}

Zhang, Xinxin ^{[3
]}

Song, Fei ^{[1
]}

Zhao, Guoping ^{[15
,17
,18
]}

Wang, Jin ^{[19
]}

机构：

[1] Shenzhen Univ, Shenzhen Univ Sch Med, Shenzhen Peoples Hosp 2, Int Canc Ctr,Affiliated Hosp 1,Dept Urol, Shenzhen 518039, Peoples R China

[2] Tongji Univ, Shanghai East Hosp, Dept Lab Med, Sch Med, Shanghai 200123, Peoples R China

[3] Shanghai Jiao Tong Univ, Ruijin Hosp, Res Lab Clin Virol, Sch Med, Shanghai 200025, Peoples R China

[4] Jinan Ctr Dis Control & Prevent, Jinan 250021, Shandong, Peoples R China

[5] Chinese Acad Sci, Shenzhen Inst Synthet Biol, Shenzhen Inst Adv Technol, Shenzhen 518055, Peoples R China

[6] Chinese Natl Human Genome Ctr Shanghai, Shanghai Most Key Lab Hlth & Dis Genom, Shanghai 201203, Peoples R China

[7] Shandong Univ, Jinan Infect Dis Hosp, Jinan 250021, Shandong, Peoples R China

[8] Shandong Univ, Med Res & Lab Diagnost Ctr, Jinan Cent Hosp, Jinan 250013, Shandong, Peoples R China

[9] Univ Sci & Technol China, Affiliated Hosp USTC 1, Div Life Sci & Med, Hefei 230001, Anhui, Peoples R China

[10] Shenzhen Ctr Dis Control & Prevent, Shenzhen 518055, Peoples R China

[11] Huazhong Univ Sci & Technol, Shenzhen Peoples Hosp 6, Nanshan Hosp, Union Shenzhen Hosp, Shenzhen 518052, Peoples R China

[12] Yuebei Second Peoples Hosp, Shaoguan 512000, Guangdong, Peoples R China

[13] Shanghai Inst Qual Inspect & Tech Res, Natl Qual Supervis & Inspect Ctr Food Prod Shangh, Shanghai 200233, Peoples R China

[14] Prov Peoples Hosp, Guangdong Acad Med Sci Guangzhou, Lab Med, Guangzhou 510080, Guangdong, Peoples R China

[15] Tolo Biotechnol Co Ltd, Shanghai 200233, Peoples R China

[16] Publ Hlth Med Ctr Chongqing Municipal, Chongqing 400036, Peoples R China

[17] Chinese Acad Sci, CAS Key Lab Synthet Biol, Inst Plant Physiol & Ecol, Shanghai Inst Biol Sci, Shanghai 200032, Peoples R China

[18] Chinese Acad Sci, CAS MPG Partner Inst Computat Biol, Key Lab Computat Biol, Biomed Big Data Ctr,Shanghai Inst Nutr & Hlth, Shanghai 200031, Peoples R China

[19] Shanghai Normal Univ, Coll Life Sci, Shanghai 200234, Peoples R China

来源：

EBIOMEDICINE | 2020年 / 61卷

基金：

中国国家自然科学基金;

关键词：

COVID-19; SARS-CoV-2; rRT-PCR; CRISPR diagnosis; Cas12a; SENA; NUCLEIC-ACID DETECTION;

D O I：

10.1016/j.ebiom.2020.103036

中图分类号：

R5 [内科学];

学科分类号：

1002 ; 100201 ;

摘要：

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 transcleavage 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. (C) 2020 The Author(s). Published by Elsevier B.V.

引用

页数：10

共 28 条

[21] Stability issues of RT-PCR testing of SARS-CoV-2 for hospitalized patients clinically diagnosed with COVID-19 [J].