Method Development and Sensitivity Optimization Paradigm for One-Step RPA-CRISPR/Cas12a Rapid Detection of the...

Area of Science:

• Molecular biology
• Antimicrobial resistance research
• Biotechnology

Background:

• Antibiotic resistance genes (ARGs) pose a significant global health threat.
• Effective monitoring methods for ARGs are crucial but face challenges in balancing sensitivity, speed, and portability.
• The carbapenem resistance gene *bla*_NDM is a key target for surveillance.

Purpose of the Study:

• To develop a rapid, sensitive, and portable one-step assay for detecting the *bla*_NDM gene.
• To establish a sensitivity optimization paradigm, MOSAIC, for RPA-CRISPR/Cas12a assays.
• To evaluate the performance of the MOSAIC assay in clinical and environmental samples.

Main Methods:

• Development of a one-step assay integrating recombinase polymerase amplification (RPA) and CRISPR/Cas12a.
• Implementation of the MOSAIC (multistrategy optimized sensitive assay via integrated CRISPR/Cas12a) paradigm for sensitivity enhancement.
• Optimization strategies included component adjustment, suboptimal-PAM-mediated CRISPR inhibition, and glycerol-assisted phase separation.

Main Results:

• The MOSAIC paradigm achieved a 10,000-fold improvement in sensitivity when combining optimization strategies.
• The assay reached a limit of detection (LOD) of 260 copies/μL for *bla*_NDM, comparable to qPCR.
• The assay provided high diagnostic sensitivity (100%) and specificity (95.45%) in clinical isolates and good accuracy in environmental samples within 1 hour at 37 °C.

Conclusions:

• The developed MOSAIC assay offers a sensitive, rapid, and potentially portable method for detecting the *bla*_NDM gene.
• The MOSAIC optimization paradigm provides a valuable framework for enhancing other one-step RPA-CRISPR/Cas12a assays.
• This technology can facilitate on-site monitoring of critical antibiotic resistance genes.