key: cord-1041781-komovd1t
authors: Chen, Yanju; Shi, Ya; Chen, Yin; Yang, Zhangnv; Wu, Hui; Zhou, Zhihui; Li, Jue; Ping, Jianfeng; He, Luping; Shen, Hong; Chen, Zhengxin; Wu, Jian; Yu, Yunsong; Zhang, Yanjun; Chen, Huan
title: Contamination-free visual detection of SARS-CoV-2 with CRISPR/Cas12a: A promising method in the point-of-care detection
date: 2020-09-20
journal: Biosens Bioelectron
DOI: 10.1016/j.bios.2020.112642
sha: 3e8f3487d96c16b3178a28248df902ac0dd47f26
doc_id: 1041781
cord_uid: komovd1t

The outbreaks of the infectious disease COVID-19 caused by SARS-CoV-2 seriously threatened the life of humans. A rapid, reliable and specific detection method was urgently needed. Herein, we reported a contamination-free visual detection method of for SARS-CoV-2 with LAMP and CRISPR/Cas12a technology. CRISPR/Cas12a reagents were pre-added on the inner wall of the tube lid. After LAMP reaction, CRISPR/Cas12a reagents were flowed into the tube and mixed with amplicon solution by hand shaking, which can effectively avoid possible amplicon formed aerosol contamination caused by re-opening the lid after amplification. CRISPR/Cas12a can highly specific recognize target sequence and discriminately cleave single strand DNA probes (5′-6FAM 3′-BHQ1). With smart phone and portable 3D printing instrument, the produced fluorescence can be seen by naked eyes without any dedicated instruments, which is promising in the point-of-care detection. The whole amplification and detection process could be completed within 40 min with high sensitivity of 20 copies RNA of SARS-CoV-2. This reaction had high specificity and could avoid cross-reactivity with other common viruses such as influenza virus. For 7 positive and 3 negative respiratory swab samples provided by Zhejiang Provincial Center for Disease Control and Prevention, our detection results had 100% positive agreement and 100% negative agreement, which demonstrated the accuracy and application prospect of this method.

A severe respiratory disease caused by SARS-CoV-2 was found in Wuhan, Hubei 61 province, China in December 2019 (Wu et al., 2020a) . The familial clustered cases 62 suggested that the virus could spread from person to person (Chan et al., 2020) .

During several weeks after the novel coronavirus were found, the virus spread rapidly 64 in China. A few months later, the virus spread around the world. As of 31st May 2020, 65 according to public data from Johns Hopkins University, more than 6 million 66 confirmed cases and 360 thousand deaths were reported in the worldwide. It was 67 found that the infectivity of SARS-CoV-2 peaked during the incubation period, which 68 may cause many clustered cases (He et al., 2020) . Diagnosis of COVID-19 in time is 69 effective for tracking and controlling the epidemic. The commonly used testing 70 methods for virus detection include immunoassays and nucleic acid detection.

However, for immunoassays, it was difficult to detect blood samples from patients in 72 the incubation and early infection period by immunoassays. Cross-reaction with other 73 similar coronaviruses may also affect the specificity of immunoassays (Lee et al., 74 2020). Therefore, immunoassays could not be used as the only basis but a supplement 75 for the diagnosis and exclusion of COVID-19. It could just be used as. qRT-PCR was 76 the gold standard for SARS-CoV-2 detection extracted from throat or nasal swabs 77 (Wu et al., 2020c) . The characteristic sequence information of SARS-CoV-2 could be 

For RT-LAMP reaction, thermo-cycling process is not required and the reaction time 88 is also shorter than qRT-PCR (Estrela et al., 2019 (Kim et al., 2020) . Based on the 221 sequencing and transcriptome analysis, we selected three gene fragments, ORF gene, 222 N gene and E gene were selected for detection (Fig. 1) . We summarized the Sequence 223 information of primers, probes and CRISPR gRNA were summarized in Table 1 

3.5 Verification the specificity of the contamination-free visual detection method 300 Highly specific detection method was important for diagnosis, which could can avoid 301 cross-reactions and improve the accuracy of detection. To demonstrate the specificity 302 of the contamination-free visual detection method, other common virus samples were 303 detected using our method. As was shown in Tab 

This greatly reduced the cost and increased the portability of testing. Among N 346 gene, E gene and ORF gene of SARS-CoV-2, we chose the ORF gene was chosen 347 with higher detection sensitivity by RT-LAMP assay. The LOD of ORF gene of 348

SARS-CoV-2 could be as low as 20 copies RNA per reaction using. The visual 349 detection process of ORF gene of SARS-CoV-2 could be

Our detection method was proved to be highly specific by testing other 17 351 common viruses. To verify the specificity of our method, we tested more than 17 352 common viruses with our method such as Conoravirus 229E

Based on the high-specificity and high-sensitivity method, 7 354 positive and 3 negative real respiratory swab samples were detected with 100% 355 positive agreement and 100% negative agreement. This fast, low-cost, high-specificity 356 and high-sensitivity method avoids using thermo-cycling instruments or specialized 357 detector and possible aerosol contamination