Photo-initiated droplet digital recombinase polymerase amplification coupled with invasive reactions enables...

Antimicrobial susceptibility testing (AST) is crucial for guiding timely antibiotic therapy. However, the conventional AST requires several days to issue a report. Although digital polymerase chain reaction (dPCR) can reduce AST turnaround time by determining bacterial susceptibility after short antibiotic exposure, the process remains time-consuming. Recombinase polymerase amplification (RPA) offers greater efficiency since it runs at room temperature and avoids amplicon denaturing or primer annealing. Nevertheless, adapting RPA to a digital format has been challenging because amplification begins prematurely before compartmentalization, inevitably leading to overestimation. Here, we proposed a photo-initiated droplet digital RPA (ddRPA) method that eliminates pre-amplification. We modified three bases within RPA primers with photocleavable groups to efficiently block RPA, and ddRPA starts only after irradiation. Due to the high efficacy of RPA in droplets, quantification is completed within only 8 min. Based on the photo-controlled strategy, we developed an ultra-fast AST platform (PIRATE) for bloodstream infections (BSI) pathogens. Using Klebsiella pneumoniae and Enterococcusfaecalis against quinolones as model systems, PIRATE demonstrates 100 % concordance with standard AST results across 33 clinical isolates and 8 blood samples while reducing the turnaround time from 3 days to under 6 h. This approach shows significant potential for guiding precise antibiotic prescription.