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Rapid and Specific Detection of Acinetobacter baumannii Infections Using a Recombinase Polymerase Amplification/Cas12a-based System
Published on: April 25, 2025
Fluorescence Encoding-Enabled Multiplexed Droplet Digital Recombinase Polymerase Amplification-CRISPR/Cas12a Assay
Yifan Wang1, Enqi Huang1, Ziyi Zhang1
1Department of Laboratory Medicine, School of Medicine, The Second Affiliated Hospital of South China University of Technology (Guangzhou First People's Hospital), Guangzhou510180, China.
Abstract:
Digital RPA-CRISPR/Cas12a assays enable accurate nucleic acid quantification; however, their multiplexing capacity is inherently limited by signal confounding arising from the promiscuous trans-cleavage activity of Cas12a effectors. To overcome this limitation, we developed a fluorescence encoding-enabled multiplexed droplet digital RPA-CRISPR/Cas12a (FLEMD-CRISPR) assay that integrates combinatorial fluorescence encoding, deterministic droplet pairing within a calabash-shaped microwell array, and a machine learning-assisted decoding pipeline. In FLEMD-CRISPR, small-volume fluorescence-encoded CRISPR/Cas12a droplets, each assigned a unique spectral signature corresponding to a specific crRNA, are one-to-one paired with large-volume RPA droplets. Following RPA amplification and droplet merging, Cas12a-mediated reporter cleavage occurs only in droplets containing target amplicons. The decoding pipeline then localizes droplets, classifies their colors to assign crRNA identities, and finally detects positive signals, enabling high-throughput, hands-off analysis of the entire droplet array. FLEMD-CRISPR enabled simultaneous quantification of nucleic acid targets from eight major lower respiratory tract infection-associated bacterial pathogens, showing strong linear correlation (R2 = 0.9987-0.9998) and consistent detection efficiency (slopes = 0.9849-1.0198) relative to single-plex digital PCR. The complete workflow, from sample preparation to final readout, can be completed within 2 h. Clinical validation using 30 bronchoalveolar lavage fluid samples showed that the bacterial detection outcomes of FLEMD-CRISPR were highly concordant with standard-of-care culture, achieving 100% (20/20) positive percent agreement and 99.1% (218/220) negative percent agreement. By enabling panel reconfiguration through simple modifications of the droplet encoding library, FLEMD-CRISPR provides a flexible platform for the development of highly multiplexed digital CRISPR assays.
