Universal CRISPR‐Cas12a and Toehold RNA Cascade Reaction on Paper Substrate for Visual Salmonella Genome Detection

Abstract Salmonella , the most prevalent food‐borne pathogen, poses significant medical and economic threats. Swift and accurate on‐site identification and serotyping of Salmonella are crucial to curb its spread and contamination. Here, we present a synthetic biology cascade reaction on a paper substrate using CRISPR‐Cas12a and recombinase polymerase amplification (RPA), enabling the programming of a standard toehold RNA switch for a genome of choice. Our approach employs just one toehold RNA switch design to differentiate between two different Salmonella serotypes, i.e., S . Typhimurium and S . Enteritidis, without the need for reengineering the toehold RNA switch. Our sensor exhibits high sensitivity, capable of visually detecting as few as 100 copies of the whole genome from a model Salmonella pathogen on a paper substrate. Furthermore, we successfully applied this robust assay to detect whole genomes in contaminated milk and lettuce samples, demonstrating its potential in real sample analysis. Due to its versatility and practical features, genomes from different organisms can be detected by merely changing a single RNA element in this universal cell‐free cascade reaction. This article is protected by copyright. All rights reserved