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Asymmetric volume-mediated buffer control overcomes sensitivity limits in one-pot RAA-CRISPR/Cas12a visual detection
Yue Zhang1,2, Zunquan Zhao2, Mingzhu Liu2
1School of Biological Science and Engineering, Shaanxi University of Technology, Hanzhong, 723000, China.
Analytical and Bioanalytical Chemistry
|September 8, 2025
Summary
This study introduces a novel, low-cost visual detection method for the colistin resistance gene mcr-1 using asymmetric volume-optimized recombinase-aided amplification (RAA) and CRISPR/Cas12a. The method enhances detection sensitivity and reduces costs for food safety applications.
Area of Science:
- Molecular Biology
- Biotechnology
- Food Safety
Background:
- Colistin resistance gene spread via the food chain necessitates rapid, low-cost, visual detection methods.
- CRISPR/Cas12a combined with recombinase-aided amplification (RAA) offers a potential one-pot, aerosol-free visual detection solution.
- Traditional one-pot RAA-CRISPR/Cas12a systems face limitations in Cas12a cleavage efficiency due to buffer incompatibility.
Purpose of the Study:
- To develop an ultrasensitive, visually detectable assay for the mobile colistin resistance gene mcr-1.
- To overcome buffer incompatibility issues in one-pot RAA-CRISPR/Cas12a systems.
- To provide a cost-effective and generalizable platform for molecular diagnostics.
Main Methods:
- An asymmetric volume-optimized RAA-CRISPR/Cas12a assay was designed, spatially segregating RAA-MIX and CRISPR/Cas12a components.
- The assay utilizes minimal reaction volumes for RAA amplification followed by automatic buffer assimilation for enhanced Cas12a trans-cleavage.
- Visual detection under UV light was employed to confirm the presence of the mcr-1 gene.
Main Results:
- The proposed method achieved ultrasensitive visual detection of the mcr-1 gene with a limit of 2.5 copies/reaction.
- A 63.1% cost reduction was observed compared to standard one-pot methods.
- The assay demonstrated high specificity, with anti-interference against other plasmids and bacteria, and satisfactory performance in detecting mcr-1 in animal-derived foods.
Conclusions:
- The asymmetric volume-optimized RAA-CRISPR/Cas12a assay provides an effective strategy for ultrasensitive and cost-efficient visual detection of mcr-1.
- Reengineering buffer microenvironments through volume asymmetry offers a generalizable approach for optimizing one-pot molecular diagnostic systems.
- This method presents a valuable tool for monitoring and curbing colistin resistance in food safety.
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