AUTHOR=Liu Shuang , Huang Siyuan , Li Fang , Sun Yuanyuan , Fu Jin , Xiao Fei , Jia Nan , Huang Xiaolan , Sun Chunrong , Zhou Juan , Wang Yi , Qu Dong 

TITLE=Rapid detection of Pseudomonas aeruginosa by recombinase polymerase amplification combined with CRISPR-Cas12a biosensing system

JOURNAL=Frontiers in Cellular and Infection Microbiology

VOLUME=Volume 13 - 2023

YEAR=2023

URL=https://www.frontiersin.org/journals/cellular-and-infection-microbiology/articles/10.3389/fcimb.2023.1239269

DOI=10.3389/fcimb.2023.1239269

ISSN=2235-2988

ABSTRACT=<p><italic>Pseudomonas aeruginosa</italic> (<italic>P. aeruginosa</italic>) is an important bacterial pathogen involved in a wide range of infections and antimicrobial resistance. Rapid and reliable diagnostic methods are of vital important for early identification, treatment, and stop of <italic>P. aeruginosa</italic> infections. In this study, we developed a simple, rapid, sensitive, and specific detection platform for <italic>P. aeruginosa</italic> infection diagnosis. The method integrated recombinase polymerase amplification (RPA) technique with clustered regularly interspaced short palindromic repeat (CRISPR)–CRISPR-associated protein 12a (Cas12a) biosensing system and was termed <italic>P. aeruginosa</italic>–CRISPR–RPA assay. The <italic>P. aeruginosa</italic>–CRISPR–RPA assay was subject to optimization of reaction conditions and evaluation of sensitivity, specificity, and clinical feasibility with the serial dilutions of <italic>P. aeruginosa</italic> genomic DNA, the non<italic>–P. aeruginosa</italic> strains, and the clinical samples. As a result, the <italic>P. aeruginosa</italic>–CRISPR–RPA assay was able to complete <italic>P. aeruginosa</italic> detection within half an hour, including RPA reaction at 42°C for 20 min and CRISPR-Cas12a detection at 37°C for 10 min. The diagnostic method exhibited high sensitivity (60 fg per reaction, ~8 copies) and specificity (100%). The results of the clinical samples by <italic>P. aeruginosa</italic>–CRISPR–RPA assay were consistent to that of the initial result by microfluidic chip method. These data demonstrated that the newly developed <italic>P. aeruginosa</italic>–CRISPR–RPA assay was reliable for <italic>P. aeruginosa</italic> detection. In summary, the <italic>P. aeruginosa</italic>–CRISPR–RPA assay is a promising tool to early and rapid diagnose <italic>P. aeruginosa</italic> infection and stop its wide spread especially in the hospital settings.</p>