Liao Yusheng, Sun Yifan, Yu Hui, Ren Jiali, He Fengjiao
State Key Laboratory of Chemo and Biosensing, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, PR China.
Hunan Key Laboratory of Forestry Edible Sources Safety and Processing, College of Food Science and Engineering, Central South University of Forestry and Technology, Changsha, Hunan 410004, PR China.
JACS Au. 2025 Jun 9;5(6):2802-2809. doi: 10.1021/jacsau.5c00379. eCollection 2025 Jun 23.
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.
核酸扩增技术在诊断中至关重要，但面临非特异性扩增和校对效率低下的挑战。基于CRISPR的方法受到切割后蛋白质持续占据的阻碍，限制了其可扩展性。在此，我们提出了一种OMEGA IsrB切口酶循环指数（ONCE）扩增方法，这是一种新型等温策略，利用RNA引导的切口酶IsrB进行位点特异性校对。ONCE独特地整合了DNA聚合酶，以从靶位点循环置换IsrB，实现高保真、一锅式指数扩增。系统验证表明，其具有阿托摩尔灵敏度和单核苷酸错配识别能力，优于CRISPR-Cas9和传统切口酶。将ONCE应用于细菌检测，可在70分钟内对铜绿假单胞菌进行定量，浓度低至4.16 CFU/mL，在临床尿液样本中与qPCR相比，灵敏度达到94.12%，特异性为100%，且无假阳性。这项工作将ONCE确立为临床和即时护理环境中精密诊断的强大、可扩展工具。
