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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; Guo, Yongkun; Wang, Chen; Yang, Zifeng; Li, Rong; Zeng, Zhiqi; Li, Hui; Zhang, Dabing; Yang, Litao.
  • Zhu Z; Joint International Research Laboratory of Metabolic and Developmental Sciences, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, 200240, PR China.
  • Guo Y; Joint International Research Laboratory of Metabolic and Developmental Sciences, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, 200240, PR China.
  • Wang C; Joint International Research Laboratory of Metabolic and Developmental Sciences, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, 200240, PR China.
  • Yang Z; State Key Laboratory of Respiratory Disease, National Clinical Research Center for Respiratory Disease, Guangzhou Institute of Respiratory Health, The First Affiliated Hospital of Guangzhou Medical University, Guangzhou, Guangdong, 510180, PR China.
  • Li R; Joint International Research Laboratory of Metabolic and Developmental Sciences, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, 200240, PR China.
  • Zeng Z; State Key Laboratory of Respiratory Disease, National Clinical Research Center for Respiratory Disease, Guangzhou Institute of Respiratory Health, The First Affiliated Hospital of Guangzhou Medical University, Guangzhou, Guangdong, 510180, PR China.
  • Li H; Zhuhai Huirui Biotechnology Co. Ltd, PR China.
  • Zhang D; Joint International Research Laboratory of Metabolic and Developmental Sciences, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, 200240, PR China.
  • Yang L; Joint International Research Laboratory of Metabolic and Developmental Sciences, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, 200240, PR China. Electronic address: yylltt@sjtu.edu.cn.
Biosens Bioelectron ; 228: 115179, 2023 May 15.
Article in English | MEDLINE | ID: covidwho-2289069
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.
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Full text: Available Collection: International databases Database: MEDLINE Main subject: Biosensing Techniques / COVID-19 Type of study: Diagnostic study / Prognostic study Limits: Humans Language: English Journal: Biosens Bioelectron Journal subject: Biotechnology Year: 2023 Document Type: Article

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Full text: Available Collection: International databases Database: MEDLINE Main subject: Biosensing Techniques / COVID-19 Type of study: Diagnostic study / Prognostic study Limits: Humans Language: English Journal: Biosens Bioelectron Journal subject: Biotechnology Year: 2023 Document Type: Article