A rapid and sensitive one-pot platform integrating fluorogenic RNA aptamers and CRISPR-Cas13a for visual detection of monkeypox virus
Biosensors and Bioelectronics(2024)
摘要
The recent global upsurge in Monkeypox virus (MPXV) outbreaks underscores the critical need for rapid and precise diagnostic solutions, particularly in resource-constrained settings. The gold standard diagnostic method, qRT-PCR, is hindered by its time-consuming nature, requirement for nucleic acid purification, expensive equipment, and the need for highly trained personnel. Traditional CRISPR/Cas fluorescence assays, relying on trans-cleavage of ssDNA/RNA reporters labeled with costly fluorophores and quenchers, pose challenges that limit their widespread application, especially for point-of-care testing (POCT). In this study, we utilized a cost-effective and stable fluorogenic RNA aptamer (Mango III), specifically binding and illuminating the fluorophore TO3-3 PEG-Biotin Fluorophore (TO3), as a reporter for Cas13a trans-cleavage activity. We propose a comprehensive strategy integrating RNA aptamer, recombinase-aided amplification (RAA), and CRISPR-Cas13a systems for the molecular detection of MPXV target. Leveraging the inherent collateral cleavage properties of the Cas13a system, we established high-sensitivity and specificity assays to distinguish MPXV from other Orthopoxviruses (OPVs). A streamlined one-pot protocol was developed to mitigate aerosol contamination risks. Our aptamer-coupled RAA-Cas13a one-pot detection method achieved a Limit of Detection (LoD) of 4 copies of target MPXV DNA in just 40 min. Validation using clinical MPX specimens confirmed the rapid and reliable application of our RAA-Cas13a-Apt assays without nucleic acid purification procedure, highlighting its potential as a point-of-care testing solution. These results underscore the user-friendliness and effectiveness of our one-pot RAA-Cas13a-Apt diagnostic platform, poised to revolutionize disease detection and management.
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关键词
Monkeypox virus,Cas13a,Aptamer,Point of care testing,One-pot detection
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