One-pot dual-CRISPR/Cas12b-mediated dUTP-LAMP for rapid and sensitive detection of foodborne pathogens without carryover contamination

Rapid and accurate detection of foodborne pathogens is of great significance for ensuring food safety. Currently, the CRISPR-Dx based on Cas12b, a new and better performing Cas enzyme, is very rare, and the vast majority require a two-step method, facing aerosol contamination. In this work, we reported a one-pot dual-CRISPR/Cas12b mediated dUTP-LAMP method for rapid and sensitive detection of foodborne pathogens without nucleic acid extraction and contamination-free. The design mechanism of sgRNAs in the one-pot CRISPR/Cas12b-mediated LAMP method was investigated in detail for the first time. Moreover, a novel PAM sequence UUN of AapCas12b was discovered, and combined with dUTP-LAMP system for one-pot dual-sgRNA detection without carryover contamination. Our developed method can achieve high specificity, nucleic acid extraction-free detection of 2.33×104 CFU/mL Salmonella within 1 h. Furthermore, accurate detection of Salmonella was successfully performed in real samples of milk, fruit and egg, demonstrating the practical significance of the developed method.