Onsite detection of airborne antibiotic-resistant bacteria via Cas9 nickase-triggered amplification reactions.

Antibiotic resistance is a critical global health issue, with methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus (VRE) being major pathogens causing pneumonia and sepsis. In this study, we introduce the Cas9 nickase-triggered amplification reaction (CN-TAR) assay - onsite, real-time detection method designed to help prevent airborne transmission of these pathogens. The assay utilizes Cas9 nickase to specifically cleave target DNA, followed by rolling circle amplification for single-step detection. To enhance filed applicability, a portable isothermal PCR device was integrated into the system. The CN-TAR assay was validated using synthetic nucleic acids, cultured bacteria, and airborne samples, achieving detection limits of 1.40 copies/μL for MRSA and 1.13 copies/μL for VRE. It demonstrated high sensitivity and rapid turnaround time. Furthermore, its performance was comparable to that of conventional reverse transcription PCR (RT-PCR), confirming its reliability for airborne antibiotic-resistant bacteria monitoring. This study presents a practical on-site detection platform, and the results highlight the CN-TAR assay as a promising tool for real-time surveillance and detection, contributing to effective infection control and public health safety.