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=Pseudomonas aeruginosa (P. aeruginosa) 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 P. aeruginosa infections. In this study, we developed a simple, rapid, sensitive and specific detection platform for P. aeruginosa infection diagnosis. The method integrated recombinase polymerase amplification (RPA) technique with CRISPR-Cas12a biosensing system and was termed P. aeruginosa-CRISPR-RPA assay. The P. aeruginosa-CRISPR-RPA assay was subject to optimization of reaction conditions and evaluation of sensitivity, specificity and clinical feasibility with the serial dilutions of P. aeruginosa genomic DNA, the non-P. aeruginosa strains and clinical samples. As a result, the P. aeruginosa-CRISPR-RPA assay was able to complete P. aeruginosa detection within half an hour, including RPA reaction at 42 ºC for 20 min and CRISPR-Cas12a detection at 37 ºC for 10 min. The diagnostic method exhibited high sensitivity (60 fg per reaction, ~8 copies) and specificity (100%). The results of clinical samples by P. aeruginosa-CRISPR-RPA assay were consistent to that of the initial result by microfluidic chip method. These data demonstrated that the newly developed P. aeruginosa-CRISPR-RPA assay was reliable for P. aeruginosa detection. In summary, the P. aeruginosa-CRISPR-RPA assay is a promising tool to early and rapid diagnose P. aeruginosa infection and stop its wide spread especially in the hospital settings.