G-Quadruplex-Probing CRISPR-Cas12 Assay for Label-Free Analysis of Foodborne Pathogens and Their Colonization In Vivo

Abstract

Foodborne pathogen infection is a key issue of food safety. Herein, we developed a label-free assay for Salmonella enterica (S. enterica) detection based on the G-quadruplex-probing CRISPR-Cas12 system (termed G-CRISPR-Cas), allowing highly sensitive detection of S. enterica and investigation of their colonization in chickens. The introduction of the G-quadruplex probe serving as the substrate of Cas 12a realized a label-free analysis for foodborne pathogens. Due to the amplification process induced by loop-mediated isothermal amplification (LAMP), G-CRISPR-Cas assay can detect S. enterica as low as 20 CFU. Specificity for pathogenic gene detection was guaranteed by the dual recognition process via LAMP primers and Cas 12a-guided RNA binding. The G-CRISPR-Cas assay was applied to explore S. enterica colonization in the intestinal tract and organs of chickens and showed the risk of S. enterica infection outside of the intestinal tract. The G-CRISPR-Cas assay is promising for on-site diagnosis of the infection or contamination of foodborne pathogens outside the laboratories, such as abattoirs and markets.

Recent Developments in G-Quadruplex Binding Ligands and Specific Beacons on Smart Fluorescent Sensor for Targeting Metal Ions and Biological Analytes

“…Subsequently, through cascade amplification, many G4s were generated and bound to NMM, resulting in fluorescence enhancement. Recently, based on the CRISPR-Cas12 system, Deng et al established a G4 sensor for analyzing foodborne pathogens’ infection and their colonization in vivo (Figure F) . The assay could be applied to facilitate the detection of Salmonella enterica and investigate its colonization in chickens.…”

“…(F) Schematic diagram of detection of foodborne pathogens and in vivo colonization by G-CRISPR-Cas assay. Reprinted with permission from ref . Copyright 2021, American Chemical Society.…”

“…Recently, based on the CRISPR-Cas12 system, Deng et al established a G4 sensor for analyzing foodborne pathogens' infection and their colonization in vivo (Figure 8F). 154 The assay could be applied to facilitate the detection of Salmonella enterica and investigate its colonization in chickens. The LAMP amplification and G-CRISPR-Cas assay were allowed for the detection S. enterica as low as 20 CFU.…”

Graphic abstract as a required element in manuscript submission and acceptance

“…G-CRISPR-Cas assay can detect S. enterica as low as 20 CFU. The analysis of extracted DNA from the pathogen was carried out with this method, and a high level of sensitivity was obtained (Xia et al, 2021).…”

Sensitive Detection of a Single-Nucleotide Polymorphism in Foodborne Pathogens Using CRISPR/Cas12a-Signaling ARMS-PCR

“…A growing number of molecular assays for Salmonella detection have thus far been exploited to meet the increasing demand for food safety. − Molecular assays such as quantitative polymerase chain reaction (qPCR) and loop-mediated isothermal amplification can produce comparable results in less time than conventional culture plating methods, especially enabling high-throughput reporting. − However, many challenges remain in developing sensitive and selective tools for single-nucleotide polymorphism (SNP) discrimination mainly due to the low abundance (<1%) of the mutated target (MT) and the minimal impact caused by SNP on the nucleic acid structure. The amplification refractory mutation system qPCR (ARMS-qPCR), an approved technology by Food and Drug Administration, has been widely used for detecting SNPs in clinical practice and exhibits higher sensitivity than DNA sequencing with a detection limit of ∼1% .…”