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<p>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 <i>trans</i>-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 <i>mcr-1</i>. 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 <i>trans</i>-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&#xa0;<i>mcr-1</i> gene was 2.5 copies/reaction, with anti-interference against other plasmids or bacteria. This method was applied to the detection of <i>mcr-1</i> 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.</p> Graphical Abstract <p></p>

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Asymmetric volume-mediated buffer control overcomes sensitivity limits in one-pot RAA-CRISPR/Cas12a visual detection

  • 期刊论文

  • 发表日期：

    2025年9月8日

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  • 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

摘要

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.

Graphical Abstract