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      CRISPR/Cas12-Based Ultra-Sensitive and Specific Point-of-Care Detection of HBV

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          Abstract

          Hepatitis B remains a major global public health challenge, with particularly high prevalence in medically disadvantaged western Pacific and African regions. Although clinically available technologies for the qPCR detection of HBV are well established, research on point-of-care testing has not progressed substantially. The development of a rapid, accurate point-of-care test is essential for the prevention and control of hepatitis B in medically disadvantaged rural areas. The development of the CRISPR/Cas system in nucleic acid detection has allowed for pathogen point-of-care detection. Here, we developed a rapid and accurate point-of-care assay for HBV based on LAMP-Cas12a. It innovatively solves the problem of point-of-care testing in 10 min, particularly the problem of sample nucleic acid extraction. Based on LAMP-Cas12a, visualization of the assay results is presented by both a fluorescent readout and by lateral flow test strips. The lateral flow test strip technology can achieve results visible to the naked eye, while fluorescence readout can achieve real-time high-sensitivity detection. The fluorescent readout-based Cas12a assay can achieve HBV detection with a limit of detection of 1 copy/μL within 13 min, while the lateral flow test strip technique only takes 20 min. In the evaluation of 73 clinical samples, the sensitivity and specificity of both the fluorescence readout and lateral flow test strip method were 100%, and the results of the assay were fully comparable to qPCR. The LAMP-Cas12a-based HBV assay relies on minimal equipment to provide rapid, accurate test results and low costs, providing significant practical value for point-of-care HBV detection.

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          Most cited references32

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          Clustal W and Clustal X version 2.0.

          The Clustal W and Clustal X multiple sequence alignment programs have been completely rewritten in C++. This will facilitate the further development of the alignment algorithms in the future and has allowed proper porting of the programs to the latest versions of Linux, Macintosh and Windows operating systems. The programs can be run on-line from the EBI web server: http://www.ebi.ac.uk/tools/clustalw2. The source code and executables for Windows, Linux and Macintosh computers are available from the EBI ftp site ftp://ftp.ebi.ac.uk/pub/software/clustalw2/
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            CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity

            CRISPR-Cas12a (Cpf1) proteins are RNA-guided enzymes that bind and cut DNA as components of bacterial adaptive immune systems. Like CRISPR-Cas9, Cas12a has been harnessed for genome editing based on its ability to generate targeted, double-stranded DNA (dsDNA) breaks. Here we show that RNA-guided DNA binding unleashes indiscriminate single-stranded DNA (ssDNA) cleavage activity by Cas12a that completely degrades ssDNA molecules. We find that target-activated, non-specific ssDNase cleavage is also a property of other type V CRISPR-Cas12 enzymes. By combining Cas12a ssDNase activation with isothermal amplification, we create a method termed DNA Endonuclease Targeted CRISPR Trans Reporter (DETECTR), which achieves attomolar sensitivity for DNA detection. DETECTR enables rapid and specific detection of human papillomavirus in patient samples, thereby providing a simple platform for molecular diagnostics.
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              CRISPR-Cas12–based detection of SARS-CoV-2

              An outbreak of betacoronavirus SARS-CoV-2 began in Wuhan, China in December 2019. COVID-19, the disease associated with infection, rapidly spread to produce a global pandemic. We report development of a rapid (<40 min), easy-to-implement and accurate CRISPR-Cas12-based lateral flow assay for detection of SARS-CoV-2 from respiratory swab RNA extracts. We validated our method using contrived reference samples and clinical samples from US patients, including 36 patients with COVID-19 infection and 42 patients with other viral respiratory infections. Our CRISPR-based DETECTR assay provides a visual and faster alternative to the US CDC SARS-CoV-2 real-time RT-PCR assay, with 95% positive predictive agreement and 100% negative predictive agreement.. SARS-CoV-2 in patient samples is detected in under an hour using a CRISPR-based lateral flow assay.
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                Author and article information

                Contributors
                Role: Academic Editor
                Journal
                Int J Mol Sci
                Int J Mol Sci
                ijms
                International Journal of Molecular Sciences
                MDPI
                1422-0067
                03 May 2021
                May 2021
                : 22
                : 9
                : 4842
                Affiliations
                [1 ]College of Public Health, Zhengzhou University, Zhengzhou 450000, China; rhd23@ 123456foxmail.com (R.D.); LJZzzu@ 123456yeah.net (J.L.); 15660129935@ 123456163.com (M.Y.); zhengxue0313@ 123456163.com (X.Z.); shenyue5151@ 123456163.com (Y.S.); jyf201907@ 123456zzu.edu.cn (Y.J.); yhy@ 123456zzu.edu.cn (H.Y.)
                [2 ]State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing 100071, China; lihao88663239@ 123456126.com
                [3 ]Key Laboratory of Molecular Medicine in Henan Province, Zhengzhou 450000, China
                Author notes
                [* ]Correspondence: sychen@ 123456zzu.edu.cn (S.C.); gcduan@ 123456zzu.edu.cn (G.D.); Tel.: +86-0371-6778-1405 (S.C. & G.D.)
                Author information
                https://orcid.org/0000-0002-1523-0808
                https://orcid.org/0000-0001-6129-0310
                Article
                ijms-22-04842
                10.3390/ijms22094842
                8125043
                34063629
                4cb64d9a-96da-408d-9e4a-d9f049f860d6
                © 2021 by the authors.

                Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ( https://creativecommons.org/licenses/by/4.0/).

                History
                : 31 March 2021
                : 29 April 2021
                Categories
                Article

                Molecular biology
                hepatitis b virus (hbv),crispr/cas12a,lamp,point-of-care detection
                Molecular biology
                hepatitis b virus (hbv), crispr/cas12a, lamp, point-of-care detection

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