Enhanced One-Pot Dual-CRISPR-Based Assay Lyophilized on a 3D-Printed Disc for Field-Deployable Multiplex Bacteria Detection.

ABSTRACT: One-pot CRISPR-based detection combining recombinase polymerase amplification (RPA) enables rapid and accurate nucleic acid testing but faces challenges in performance, multiplexing, and field-ready lyophilization. Here, an enhanced one-pot, helicase-assisted RPA (hRPA)-combined, dual-CRISPR/uAsCas12a (EOD-CRISPR) assay is described which can be lyophilized on a 3D-printed microfluidic disc to achieve field-deployable multiplex bacteria detection. In EOD-CRISPR reactions, the reaction speed, sensitivity, and fluorescence signal are significantly enhanced due to the synergistic effect of bovine serum albumin, hRPA, and uAsCas12a nuclease. The 3D-printed disc features four central chambers encircled by eight outer chambers, permitting detecting four targets simultaneously. For stable lyophil

SUBMITTER: Shen Y 

PROVIDER: S-EPMC12677630 | biostudies-literature | 2025 Dec

REPOSITORIES: biostudies-literature