Rapid and visual one-tube detection of Pseudomonas tolaasii based on recombinase-aided amplification and CRISPR/Cas12a

Pseudomonas tolaasii , a widely distributed pathogenic bacterium, causes brown blotch disease of edible mushrooms, limiting the advancement of the edible mushroom industry in China. A one-tube method platform that combined clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) system and recombinase-aided amplification (RAA) for P. tolaasii detection was developed. The sub-optimal protospacer adjacent motif (PAM) sequence was selected for the recognition of target DNA to achieve the one-tube detection. Additionally, fluorescent DNA probe and lateral-flow dipsticks (LFD) were utilized for visualization. The established one-tube detection method enabled visual detection, achieving detection limits of 1.6 × 10 −6  ng/μL and 1.6 × 10 −5  ng/μL P. tolaasii recombined plasmid, respectively, by integrating LFD or fluorescent ssDNA probe. It exhibited good specificity, successfully recognizing the P. tolaasii from other 5 common bacteria, including P. aeruginosa , Staphylococcus aureus , Escherichia coli , Brevundimonas di min uta , and Bacillus thuringiensis. The detection time was approximately 25 min. Overall, a CRISPR-coupled RAA method employing suboptimal PAM sequences was developed for one-tube, visual detection of P. tolaasii with high sensitivity and specificity, enabling visual naked-eye readout. The developed method offers a convenient and user-friendly tool for the early diagnosis of P. tolaasii -related diseases in edible mushroom cultivation.