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An ultra-sensitive one-pot RNA-templated DNA ligation rolling circle amplification-assisted CRISPR/Cas12a detector assay for rapid detection of SARS-CoV-2.


ABSTRACT: Rapid, sensitive, and one-pot diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) plays an extremely important role in point-of-care testing (POCT). Herein, we report an ultra-sensitive and rapid one-pot enzyme-catalyzed rolling circle amplification-assisted CRISPR/FnCas12a assay, termed OPERATOR. OPERATOR employs a single well-designed single-strand padlock DNA, containing a protospacer adjacent motif (PAM) site and a sequence complementary to the target RNA which procedure converts and amplifies genomic RNA to DNA by RNA-templated DNA ligation and multiply-primed rolling circle amplification (MRCA). The MRCA amplicon of single-stranded DNA is cleaved by the FnCas12a/crRNA complex and detected via a fluorescence reader or lateral flow strip. OPERATOR presents outstanding advantages including ultra-sensitivity (1.625 copies per reaction), high specificity (100%), rapid reaction speed (∼30 min), easy operation, low cost, and on-spot visualization. Furthermore, we established a POCT platform by combining OPERATOR with rapid RNA release and a lateral flow strip without professional equipment. The high performance of OPERATOR in SARS-CoV-2 tests was confirmed using both reference materials and clinical samples, and the results suggest that is readily adaptable for point-of-care testing of other RNA viruses.

SUBMITTER: Zhu Z 

PROVIDER: S-EPMC9974209 | biostudies-literature | 2023 May

REPOSITORIES: biostudies-literature

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An ultra-sensitive one-pot RNA-templated DNA ligation rolling circle amplification-assisted CRISPR/Cas12a detector assay for rapid detection of SARS-CoV-2.

Zhu Zaobing Z   Guo Yongkun Y   Wang Chen C   Yang Zifeng Z   Li Rong R   Zeng Zhiqi Z   Li Hui H   Zhang Dabing D   Yang Litao L  

Biosensors & bioelectronics 20230301


Rapid, sensitive, and one-pot diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) plays an extremely important role in point-of-care testing (POCT). Herein, we report an ultra-sensitive and rapid one-pot enzyme-catalyzed rolling circle amplification-assisted CRISPR/FnCas12a assay, termed OPERATOR. OPERATOR employs a single well-designed single-strand padlock DNA, containing a protospacer adjacent motif (PAM) site and a sequence complementary to the target RNA which procedur  ...[more]

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