Sensitive and Direct Analysis of Pseudomonas aeruginosa through Self-Primer-Assisted Chain Extension and CRISPR-Cas12a-Based Color Reaction.

Abstract

Pseudomonas aeruginosa (P. aeruginosa) is a common opportunistic Gram-negative pathogen that may cause infections to immunocompromised patients. However, sensitive and reliable analysis of P. aeruginosa remains a huge challenge. In this method, target recognition assists the formation of a self-primer and initiates single-stranded chain production. The produced single-stranded DNA chain is identified by CRISPR-Cas12a, and consequently, the trans-cleavage activity of the Cas12a enzyme is activated to parallelly digest Ag+ aptamer sequences that are chelated with silver ions (Ag+). The released Ag+ reacted with 3,3',5,5'-tetramethylbenzidine (TMB) for coloring. Compared with the traditional color developing strategies, which mainly rely on the DNA hybridization, the color developing strategy in this approach exhibits a higher efficiency due to the robust trans-cleavage activity of the Cas12a enzyme. Consequently, the method shows a low limit of detection of a wide detection of 5 orders of magnitudes and a low limit of detection of 21 cfu/mL, holding a promising prospect in early diagnosis of infections. Herein, we develop a sensitive and reliable method for direct and colorimetric detection of P. aeruginosa by integrating self-primer-assisted chain production and CRISPR-Cas12a-based color reaction and believe that the established approach will facilitate the development of bacteria-analyzing sensors.