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

被引:37
作者
Zhu, Zaobing [1 ]
Guo, Yongkun [1 ]
Wang, Chen [1 ]
Yang, Zifeng [2 ]
Li, Rong [1 ]
Zeng, Zhiqi [2 ]
Li, Hui [3 ]
Zhang, Dabing [1 ]
Yang, Litao [1 ]
机构
[1] Shanghai Jiao Tong Univ, Sch Life Sci & Biotechnol, Joint Int Res Lab Metab & Dev Sci, Shanghai 200240, Peoples R China
[2] Guangzhou Med Univ, Guangzhou Inst Resp Hlth, Natl Clin Res Ctr Resp Dis, State Key Lab Resp Dis,Affiliated Hosp 1, Guangzhou 510180, Guangdong, Peoples R China
[3] Zhuhai Huirui Biotechnol Co Ltd, Zhuhai, Peoples R China
关键词
OPERATOR; SARS-CoV-2; CRISPR/Cas12a; RCA; RNA-Templated DNA ligation;
D O I
10.1016/j.bios.2023.115179
中图分类号
Q6 [生物物理学];
学科分类号
071011 ;
摘要
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 (similar to 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.
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页数:12
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