Isothermal one-pot RPA-CRISPR/Cas12b assay for rapid and highly sensitive detection of Brucella spp

As a zoonotic disease caused by Brucella species, brucellosis requires rapid, sensitive, and precise diagnostic approaches to effectively curb its epidemiological spread. Herein, we developed a novel isothermal one-pot recombinase polymerase amplification (RPA)-CRISPR/Cas12b (IOPR-Cas12b) assay targeting the conserved region of the Brucella spp.-specific BCSP31 gene sequence. This assay is integrated with real-time fluorescence detection and lateral flow biosensor (LFB), recognized as promising tools for Point-of-care testing (POCT) molecular diagnostics. A BCSP31-targeted IOPR-Cas12b assay was developed through systematic design and screening of RPA primers and guide RNA (gRNA), and optimization of reaction conditions. The IOPR-Cas12b achieved maximum efficiency at 43 °C for 40 min with real-time fluorescence and LFB readout methods. The system exhibited remarkable sensitivity with limits of detection (LOD) of 3.65 × 10¹ copies/reaction (real-time fluorescence) and 3.65 × 10² copies/reaction (LFB), while maintaining strict specificity by showing no cross-reactivity with 19 non-target bacterial species. Clinical validation using 61 samples demonstrated 100 % concordance between the IOPR-Cas12b assay and reference methods (culture and conventional PCR). The entire detection workflow was accomplished within 40 min through real-time fluorescence monitoring. Furthermore, the incorporation of visual LFB detection eliminated the reliance on sophisticated instrumentation, enhancing its suitability for field-deployable POCT applications. The developed one-pot platform combining IOPR-Cas12b with dual-mode detection (real-time fluorescence/LFB) offers a rapid, sensitive, and highly specific solution, providing a highly competitive technical means for brucellosis screening and prevention.