LAUSR

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 lyophilization on disc chambers, glassfiber membranes are inserted as substrates to adsorb the EOD‐CRISPR reagents containing a protectant of 5% trehalose and 1% glycine. Toward point‐of‐need testing, EOD‐CRISPR‐lyophilized discs are applied to build an onsite detection platform. Through detecting synthetic food samples contaminated by four foodborne bacteria (i.e., Bacillus cereus, Salmonella enterica, Staphylococcus aureus, and Escherichia coli O157:H7), the onsite detection platform is validated and the sensitivity (80%–88.9%) and specificity (92.3%–100%) are comparable to those of standard PCR methods. Therefore, the field‐deployable multiplex EOD‐CRISPR assays holds great potentials for onsite bacteria detection and beyond.