Enzyme-mediated "one to more" PAM-independent and amplification free CRISPR/Cas12a for one-pot rapid detection of food contaminated bacteria.

The requirement of protospacer-adjacent motif (PAM) and nucleic acid amplification have restricted the application of CRISPR/Cas12a system in bacteria detection. Herein, we proposed a restriction endonuclease mediated, "one to more", PAM-independent and amplification-free CRISPR/Cas12a strategy for bacteria detection. The bacterial genomic DNA can be cleaved by specific endonuclease, exposing a large number of sticky ends. The combination of these exposed sticky ends with a short ssDNA could effectively activate the trans-cleavage activity of Cas12a to break a FQ reporter probe, achieving the purpose of detection. This approach not only overcomes the dependence of PAM sequence but also enables one-pot detection of target bacteria within 60 min without the requirement of nucleic acid amplification. The detection sensitivity of two typical food contaminated bacteria, Alicyclobacillus acidoterrestris and Cronobacter sakazakii was 13.38 CFU/mL and 18.67 CFU/mL, respectively. In summary, this work provided a novel strategy and powerful tool for bacteria analysis and molecular diagnosis.