Yue Zhang, Zunquan Zhao, Mingzhu Liu, Jincai Yang, Chun Yang, Nan Su, Jingran Sun, Yanjun Fang, Yonghui Wang, Xiaoli Li, Wang Chen, Jin Wu, Jialei Bai
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引用次数: 0
Abstract
Rapid, low-cost, and visual nucleic acid detection methods are highly attractive for curbing colistin resistance spread through the food chain. CRISPR/Cas12a combined with recombinase-aided amplification (RAA) offers a one-pot, aerosol-free approach for visual detection. However, traditional one-pot systems often run Cas12a trans-cleavage in a buffer suitable for RAA, thus limiting Cas12a cleavage efficiency. This study proposes an asymmetric volume-optimized RAA-CRISPR/Cas12a assay for ultrasensitive visual detection of mobile colistin resistance gene mcr-1. Unlike conventional one-pot systems constrained by buffer incompatibility, our design spatially segregates a minimal-volume RAA-MIX (lid) from a CRISPR-dominant buffer microenvironment (tube bottom). This architecture leverages RAA's exponential amplification power to ensure sufficient product yield from minimal reaction volumes, while enabling subsequent enhancement of Cas12a trans-cleavage through automatic buffer assimilation upon mixing. The results were able to be visually observed under UV light, achieving 63.1% cost reduction compared to standard one-pot methods. The sensitivity of the proposed method for the mcr-1 gene was 2.5 copies/reaction, with anti-interference against other plasmids or bacteria. This method was applied to the detection of mcr-1 in animal-derived foods, showing satisfactory practical performance. By fundamentally reengineering buffer microenvironments through volume asymmetry, this work provides a general strategy for one-pot molecular diagnostics, achieving dual optimization of amplification and cleavage without trade-offs.
期刊介绍:
Analytical and Bioanalytical Chemistry’s mission is the rapid publication of excellent and high-impact research articles on fundamental and applied topics of analytical and bioanalytical measurement science. Its scope is broad, and ranges from novel measurement platforms and their characterization to multidisciplinary approaches that effectively address important scientific problems. The Editors encourage submissions presenting innovative analytical research in concept, instrumentation, methods, and/or applications, including: mass spectrometry, spectroscopy, and electroanalysis; advanced separations; analytical strategies in “-omics” and imaging, bioanalysis, and sampling; miniaturized devices, medical diagnostics, sensors; analytical characterization of nano- and biomaterials; chemometrics and advanced data analysis.