tetano
Editor, Senior Moderator
Biosens Bioelectron
. 2023 Mar 1;228:115179.
doi: 10.1016/j.bios.2023.115179. Online ahead of print.
An ultra-sensitive one-pot RNA-templated DNA ligation rolling circle amplification-assisted CRISPR/Cas12a detector assay for rapid detection of SARS-CoV-2
Zaobing Zhu[SUP] 1 [/SUP], Yongkun Guo[SUP] 1 [/SUP], Chen Wang[SUP] 1 [/SUP], Zifeng Yang[SUP] 2 [/SUP], Rong Li[SUP] 1 [/SUP], Zhiqi Zeng[SUP] 2 [/SUP], Hui Li[SUP] 3 [/SUP], Dabing Zhang[SUP] 1 [/SUP], Litao Yang[SUP] 4 [/SUP]
Affiliations
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.
Keywords: CRISPR/Cas12a; OPERATOR; RCA; RNA-Templated DNA ligation; SARS-CoV-2.
. 2023 Mar 1;228:115179.
doi: 10.1016/j.bios.2023.115179. Online ahead of print.
An ultra-sensitive one-pot RNA-templated DNA ligation rolling circle amplification-assisted CRISPR/Cas12a detector assay for rapid detection of SARS-CoV-2
Zaobing Zhu[SUP] 1 [/SUP], Yongkun Guo[SUP] 1 [/SUP], Chen Wang[SUP] 1 [/SUP], Zifeng Yang[SUP] 2 [/SUP], Rong Li[SUP] 1 [/SUP], Zhiqi Zeng[SUP] 2 [/SUP], Hui Li[SUP] 3 [/SUP], Dabing Zhang[SUP] 1 [/SUP], Litao Yang[SUP] 4 [/SUP]
Affiliations
- PMID: 36878066
- DOI: 10.1016/j.bios.2023.115179
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.
Keywords: CRISPR/Cas12a; OPERATOR; RCA; RNA-Templated DNA ligation; SARS-CoV-2.