Filtration-based LAMP-CRISPR/Cas12a system for the rapid, sensitive and visualized detection of Escherichia coli O157:H7

Abstract

Escherichia coli O157:H7 is a major cause of fresh vegetable-associated infections that can threaten human health. A method for rapidly detecting food-borne pathogens should be developed for safe food management. A clustered regularly interspaced short palindromic repeats (CRISPR)-based detection method has the potential to greatly advance biosensing technology through its high sensitivity and specificity. In this study, we developed a rapid, sensitive, and visualized method of detecting E. coli O157:H7 (stx2 gene) based on a loop-mediated isothermal amplification (LAMP)-CRISPR/Cas12a system. The developed method was able to rectify the common false-negative results produced by LAMP, and the detection limit was 1.22 × 10⁰ CFU/mL in pure culture. Furthermore, the LAMP-CRISPR/Cas12a system using filtration enrichment successfully detected 4.80 × 10⁰ CFU/g of E. coli O157:H7 in romaine lettuce without pre-microbial enrichment culture. Consequently, the LAMP-CRISPR/Cas12a system is a useful technique for rapid and sensitive detection of E. coli O157:H7 in fresh products.

Keywords: CRISPR/Cas12a; Escherichia coli O157:H7; Filtration; Rapid detection; Visualized LAMP system.