CRISPR/Cas13a analysis based on NASBA amplification for norovirus detection.

Autor: Mao Z; School of Health Science and Engineering, University of Shanghai for Science and Technology, Shanghai, 200093, China; Tianjin Key Laboratory of Risk Assessment and Control Technology for Environment and Food Safety, Tianjin Institute of Environmental and Operational Medicine, Tianjin, 300050, China., Lei H; School of Health Science and Engineering, University of Shanghai for Science and Technology, Shanghai, 200093, China., Chen R; Tianjin Key Laboratory of Risk Assessment and Control Technology for Environment and Food Safety, Tianjin Institute of Environmental and Operational Medicine, Tianjin, 300050, China., Ren S; Tianjin Key Laboratory of Risk Assessment and Control Technology for Environment and Food Safety, Tianjin Institute of Environmental and Operational Medicine, Tianjin, 300050, China. Electronic address: renshuyue2018@163.com., Liu B; School of Health Science and Engineering, University of Shanghai for Science and Technology, Shanghai, 200093, China. Electronic address: blliuk@163.com., Gao Z; Tianjin Key Laboratory of Risk Assessment and Control Technology for Environment and Food Safety, Tianjin Institute of Environmental and Operational Medicine, Tianjin, 300050, China. Electronic address: gaozhx@163.com.
Jazyk: angličtina
Zdroj: Talanta [Talanta] 2024 Dec 01; Vol. 280, pp. 126725. Date of Electronic Publication: 2024 Aug 20.
DOI: 10.1016/j.talanta.2024.126725
Abstrakt: Human norovirus (HuNoV) is a leading cause of foodborne diseases worldwide, making rapid and accurate detection crucial for prevention and control. In recent years, the CRISPR/Cas13a system, known for its single-base resolution in RNA recognition and unique collateral cleavage activity, is particularly suitable for sensitive and rapid RNA detection. However, isothermal amplification-based CRISPR/Cas13 assays often require an external transcription step, complicating the detection process. In our study, an efficient diagnostic technique based on the NASBA/Cas13a system was established to identify conserved regions at the ORF1-ORF2 junction of norovirus. The RNA amplification techniques [Nucleic Acid Sequence-Based Amplification (NASBA)] integrates reverse transcription and transcription steps, enabling sensitive, accurate, and rapid enrichment of low-abundance RNA. Furthermore, the CRISPR/Cas13a system provides secondary precise recognition of the amplified products, generating a fluorescence signal through its activated accessory collateral cleavage activity. We optimized the reaction kinetics parameters of Cas13a and achieved a detection limit as low as 51pM. The conditions for the cascade reaction involving CRISPR analysis and RNA amplification were optimized. Finally, we validated the reliability and accuracy of the NASBA/Cas13a method by detecting norovirus in shellfish, achieving results comparable to qRT-PCR in a shorter time and detecting viral loads as low as 10 copies/μL.
Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
(Copyright © 2024 Elsevier B.V. All rights reserved.)
Databáze: MEDLINE
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