High-Fidelity, One-Pot Nucleic Acid Amplification via OMEGA IsrB Nickase Cycling for Clinical Pathogen Detection.

Nucleic acid amplification technologies are pivotal in diagnostics but face challenges from nonspecific amplification and inefficient proofreading. CRISPR-based methods are hindered by persistent protein occupation postcleavage, limiting scalability. Here, we present an OMEGA IsrB Nickase Cyclic Exponential (ONCE) amplification, a novel isothermal strategy leveraging the RNA-guided nickase IsrB for site-specific proofreading. ONCE uniquely integrates DNA polymerase to cyclically displace IsrB from target sites, enabling high-fidelity, one-pot exponential amplification. Systematic validation demonstrates attomolar sensitivity and single-nucleotide mismatch discrimination, outperforming those of CRISPR-Cas9 and conventional nickases. Applied to bacterial detection, ONCE quantifies Pseudomonas aeruginosa at 4.16 CFU/mL within 70 min, achieving 94.12% sensitivity and 100% specificity in clinical urine samples with no false-positives compared to qPCR. This work establishes ONCE as a robust, scalable tool for precision diagnostics in clinical and point-of-care settings.