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Fast and visual detection of nucleic acids using a one-step RPA-CRISPR detection (ORCD) system unrestricted by the PAM.
Lin, Kangfeng; Guo, Jianguang; Guo, Xiangju; Li, Qinghan; Li, Xiao; Sun, Zhen; Zhao, Zehang; Weng, Jiao; Wu, Jinzhun; Zhang, Rui; Li, Boan.
  • Lin K; State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Signaling Network and Engineering Research Center of Molecular Diagnostics of the Ministry of Education, School of Life Sciences, Xiamen University, Xiamen, Fujian, China.
  • Guo J; State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Signaling Network and Engineering Research Center of Molecular Diagnostics of the Ministry of Education, School of Life Sciences, Xiamen University, Xiamen, Fujian, China.
  • Guo X; State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Signaling Network and Engineering Research Center of Molecular Diagnostics of the Ministry of Education, School of Life Sciences, Xiamen University, Xiamen, Fujian, China.
  • Li Q; State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Signaling Network and Engineering Research Center of Molecular Diagnostics of the Ministry of Education, School of Life Sciences, Xiamen University, Xiamen, Fujian, China.
  • Li X; State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Signaling Network and Engineering Research Center of Molecular Diagnostics of the Ministry of Education, School of Life Sciences, Xiamen University, Xiamen, Fujian, China.
  • Sun Z; State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Signaling Network and Engineering Research Center of Molecular Diagnostics of the Ministry of Education, School of Life Sciences, Xiamen University, Xiamen, Fujian, China.
  • Zhao Z; State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Signaling Network and Engineering Research Center of Molecular Diagnostics of the Ministry of Education, School of Life Sciences, Xiamen University, Xiamen, Fujian, China.
  • Weng J; State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Signaling Network and Engineering Research Center of Molecular Diagnostics of the Ministry of Education, School of Life Sciences, Xiamen University, Xiamen, Fujian, China.
  • Wu J; Women and Children's Hospital, School of Medicine, Xiamen University, NO.10 Zhenhai Road, Siming District, Xiamen, Fujian, 361003, China. Electronic address: 1923731201@qq.com.
  • Zhang R; Xiamen Cell Therapy Research Center, The First Affiliated Hospital of Xiamen University, School of Medicine, Xiamen University, Xiamen, 361003, China. Electronic address: raissarui@foxmail.com.
  • Li B; State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Signaling Network and Engineering Research Center of Molecular Diagnostics of the Ministry of Education, School of Life Sciences, Xiamen University, Xiamen, Fujian, China. Electronic address: bali@xmu.edu.cn.
Anal Chim Acta ; 1248: 340938, 2023 Apr 01.
Article in English | MEDLINE | ID: covidwho-2242732
ABSTRACT
CRISPR-Cas12a (Cpf1) is widely used for pathogen detection. However, most Cas12a nucleic acid detection methods are limited by a PAM sequence requirement. Moreover, preamplification and Cas12a cleavage are separate. Here, we developed a one-step RPA-CRISPR detection (ORCD) system unrestricted by the PAM sequence with high sensitivity and specificity that offers one-tube, rapid, and visually observable detection of nucleic acids. In this system, Cas12a detection and RPA amplification are performed simultaneously, without separate preamplification and product transfer steps, and 0.2 copies/µL of DNA and 0.4 copies/µL of RNA can be detected. In the ORCD system, the activity of Cas12a is the key to the nucleic acid detection; specifically, reducing Cas12a activity increases the sensitivity of ORCD assay detection of the PAM target. Furthermore, by combining this detection technique with a nucleic acid extraction-free method, our ORCD system can be used to extract, amplify and detect samples within 30 min, as verified with tests of 82 Bordetella pertussis clinical samples with a sensitivity and specificity of 97.30% and 100% compared with PCR. We also tested 13 SARS-CoV-2 samples with RT-ORCD, and the results were consistent with RT-PCR.
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Full text: Available Collection: International databases Database: MEDLINE Main subject: Nucleic Acids / COVID-19 Type of study: Diagnostic study / Prognostic study Limits: Humans Language: English Journal: Anal Chim Acta Year: 2023 Document Type: Article Affiliation country: J.aca.2023.340938

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Full text: Available Collection: International databases Database: MEDLINE Main subject: Nucleic Acids / COVID-19 Type of study: Diagnostic study / Prognostic study Limits: Humans Language: English Journal: Anal Chim Acta Year: 2023 Document Type: Article Affiliation country: J.aca.2023.340938