A one-step, one-pot CRISPR nucleic acid detection platform (CRISPR-top): Application for the diagnosis of COVID-19.

Li, S; Huang, J; Ren, L; Jiang, W; Wang, M; Zhuang, L; Zheng, Q; Yang, R; Zeng, Y; Luu, LDW; Wang, Y; Tai, J

A one-step, one-pot CRISPR nucleic acid detection platform (CRISPR-top): Application for the diagnosis of COVID-19.

Li, S Huang, J Ren, L Jiang, W Wang, M Zhuang, L Zheng, Q Yang, R Zeng, Y Luu, LDW Wang, Y Tai, J

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Publisher:: Elsevier BV
Publication Type:: Journal Article
Citation:: Talanta, 2021, 233, pp. 122591
Issue Date:: 2021-10-01

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Field	Value	Language
dc.contributor.author	Li, S
dc.contributor.author	Huang, J
dc.contributor.author	Ren, L
dc.contributor.author	Jiang, W
dc.contributor.author	Wang, M
dc.contributor.author	Zhuang, L
dc.contributor.author	Zheng, Q
dc.contributor.author	Yang, R
dc.contributor.author	Zeng, Y
dc.contributor.author	Luu, LDW
dc.contributor.author	Wang, Y
dc.contributor.author	Tai, J
dc.date.accessioned	2022-04-05T20:34:36Z
dc.date.available	2021-06-04
dc.date.available	2022-04-05T20:34:36Z
dc.date.issued	2021-10-01
dc.identifier.citation	Talanta, 2021, 233, pp. 122591
dc.identifier.issn	0039-9140
dc.identifier.issn	1873-3573
dc.identifier.uri	http://hdl.handle.net/10453/155949
dc.description.abstract	The existing CRISPR-mediated diagnostic tests require a two-step procedure (DNA or RNA amplification followed by CRISPR-mediated sequence-specific detection) for nucleic acid detection, which increases complexity and the risk of sample cross-contamination. Here, we report a new CRISPR-mediated test, called CRISPR-top (CRISPR-mediated testing in one-pot), which integrates simultaneous target pre-amplification with CRISPR/cas12b-mediated detection into a one-pot reaction mixture, performed at a constant temperature. The novel CRISPR-top assay was applied to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent for COVID-19 (coronavirus disease 2019). COVID-19 CRISPR-top targets the ORF1ab (opening reading frame 1a/b) and NP (nucleoprotein) genes of SARS-CoV-2, and operates at 59 °C for 40 min with minimal instrument. The COVID-19 CRISPR-top assay can return results within 60-min and is easily interpreted by visual fluorescence or lateral flow readouts. The analytical limit of detection (LoD) for COVID-19 CRISPR-top is 10 copies (for each detection target) per reaction with no cross-reactivity observed from non-SARS-CoV-2 templates. Among clinically collected non-COVID-19 samples, the assay's specificity was 100% (80/80 oropharynx swab samples). Among 52 COVID-19 positive clinical samples collected, the COVID-19 CRISPR-top assay yielded 38 (73.1%) positive results using fluorescence readout and 35 (67.3%) positive results with lateral-flow readout. These diagnostic results were similar to those obtained using RT-PCR (34 positive (65.4%)). These data indicate that COVID-19 CRISPR-top is a simple, rapid, accurate and highly sensitive method for SARS-CoV-2 detection which can be used in the clinic, field laboratories and primary care facilities in resource-challenged settings.
dc.format	Print-Electronic
dc.language	eng
dc.publisher	Elsevier BV
dc.relation.ispartof	Talanta
dc.relation.isbasedon	10.1016/j.talanta.2021.122591
dc.rights	info:eu-repo/semantics/openAccess
dc.subject	0301 Analytical Chemistry, 0399 Other Chemical Sciences
dc.subject.classification	Analytical Chemistry
dc.subject.mesh	COVID-19
dc.subject.mesh	CRISPR-Cas Systems
dc.subject.mesh	Clustered Regularly Interspaced Short Palindromic Repeats
dc.subject.mesh	Humans
dc.subject.mesh	Nucleic Acid Amplification Techniques
dc.subject.mesh	RNA, Viral
dc.subject.mesh	SARS-CoV-2
dc.subject.mesh	Sensitivity and Specificity
dc.subject.mesh	Humans
dc.subject.mesh	RNA, Viral
dc.subject.mesh	Sensitivity and Specificity
dc.subject.mesh	Nucleic Acid Amplification Techniques
dc.subject.mesh	CRISPR-Cas Systems
dc.subject.mesh	Clustered Regularly Interspaced Short Palindromic Repeats
dc.subject.mesh	COVID-19
dc.subject.mesh	SARS-CoV-2
dc.title	A one-step, one-pot CRISPR nucleic acid detection platform (CRISPR-top): Application for the diagnosis of COVID-19.
dc.type	Journal Article
utslib.citation.volume	233
utslib.location.activity	Netherlands
utslib.for	0301 Analytical Chemistry
utslib.for	0399 Other Chemical Sciences
pubs.organisational-group	/University of Technology Sydney
pubs.organisational-group	/University of Technology Sydney/Faculty of Science
pubs.organisational-group	/University of Technology Sydney/Faculty of Science/School of Life Sciences
utslib.copyright.status	open_access	*
dc.date.updated	2022-04-05T20:34:30Z
pubs.publication-status	Published
pubs.volume	233

Abstract:

The existing CRISPR-mediated diagnostic tests require a two-step procedure (DNA or RNA amplification followed by CRISPR-mediated sequence-specific detection) for nucleic acid detection, which increases complexity and the risk of sample cross-contamination. Here, we report a new CRISPR-mediated test, called CRISPR-top (CRISPR-mediated testing in one-pot), which integrates simultaneous target pre-amplification with CRISPR/cas12b-mediated detection into a one-pot reaction mixture, performed at a constant temperature. The novel CRISPR-top assay was applied to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent for COVID-19 (coronavirus disease 2019). COVID-19 CRISPR-top targets the ORF1ab (opening reading frame 1a/b) and NP (nucleoprotein) genes of SARS-CoV-2, and operates at 59 °C for 40 min with minimal instrument. The COVID-19 CRISPR-top assay can return results within 60-min and is easily interpreted by visual fluorescence or lateral flow readouts. The analytical limit of detection (LoD) for COVID-19 CRISPR-top is 10 copies (for each detection target) per reaction with no cross-reactivity observed from non-SARS-CoV-2 templates. Among clinically collected non-COVID-19 samples, the assay's specificity was 100% (80/80 oropharynx swab samples). Among 52 COVID-19 positive clinical samples collected, the COVID-19 CRISPR-top assay yielded 38 (73.1%) positive results using fluorescence readout and 35 (67.3%) positive results with lateral-flow readout. These diagnostic results were similar to those obtained using RT-PCR (34 positive (65.4%)). These data indicate that COVID-19 CRISPR-top is a simple, rapid, accurate and highly sensitive method for SARS-CoV-2 detection which can be used in the clinic, field laboratories and primary care facilities in resource-challenged settings.

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http://hdl.handle.net/10453/155949