A three-way junction probe triggered CRISPR/Cas14a1 enhanced EXPonential amplification reaction for sensitive Pseudomonas aeruginosa detection

Abstract

Pseudomonas aeruginosa (P. aeruginosa, PA) is a rod-shaped Gram-negative opportunistic bacterium capable of causing nosocomial infections during nursing, such as burn wound infections and meningitis. However, sensitive and rapid PA detection remains a huge challenge. Herein, a new fluorescent biosensor was developed for the sensitive detection of PA using a three-way junction (TWJ) probe for specific identification and CRISPR/Cas14a1 for enhanced exponential amplification. The interaction between PA and its aptamer on a DNA TWJ structure probe triggered the migration of the double-stranded DNA branch, inducing DNA polymerase/endonuclease assisted chain displacement and the generation of single-stranded DNA sequences. The amplification products have the ability to activate CRISPR/Cas14a1, resulting in effective trans-cleavage and the subsequent release of fluorescence from the reporter probes. Under optimized conditions, the proposed biosensor was able to detect a wide range of bacterial concentrations, ranging from 10 to 10⁵ cfu mL⁻¹ within 30 min. The limit of detection, which is the lowest concentration that can be reliably detected, was determined to be 3.4 cfu mL⁻¹ according to the 3δ rule. The results of the recovery test suggest that the biosensor shows significant potential for clinical applications. The established biosensor utilizing the TWJ probe generated multiple isothermal exponential amplification and the CRISPR/Cas14a1 biosensor is an excellent platform for rapidly detecting pathogenic bacteria in postoperative infection.