Biosensors & bioelectronicsMay 19, 2026

Rapid one-step detection of food bacteria using enzyme-driven CRISPR/Cas12a without signal amplification or PAM requirement

CRISPR Gene Editing Weekly Brief ↗PubMed ↗DOI ↗

Updated Jun 27, 2026

Abstract

Essence

A restriction-enzyme CRISPR/Cas12a strategy enabled one-pot bacterial DNA detection without PAM dependence or amplification.

Evidence

This molecular detection platform experiment tested food-contaminating bacteria detection for Alicyclobacillus acidoterrestris and Cronobacter sakazakii, reaching 13.38 CFU/mL and 18.67 CFU/mL sensitivity within 60 minutes.

Caveat

The work validates an assay strategy for selected bacteria, not real-world clinical or food-safety deployment across diverse samples.

Simplified

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.