Ultrasensitive and Rapid Visual Detection of Escherichia coli O157:H7 Based on RAA-CRISPR/Cas12a System

Abstract

Escherichia coli (E. coli) O157:H7 is a major foodborne and waterborne pathogen that can threaten human health. Due to its high toxicity at low concentrations, it is crucial to establish a time-saving and highly sensitive in situ detection method. Herein, we developed a rapid, ultrasensitive, and visualized method for detecting E. coli O157:H7 based on a combination of Recombinase-Aided Amplification (RAA) and CRISPR/Cas12a technology. The CRISPR/Cas12a-based system was pre-amplified using the RAA method, which showed high sensitivity and enabled detecting as low as ~1 CFU/mL (fluorescence method) and 1 × 102 CFU/mL (lateral flow assay) of E. coli O157:H7, which was much lower than the detection limit of the traditional real-time PCR technology (103 CFU/mL) and ELISA (104~107 CFU/mL). In addition, we demonstrated that this method still has good applicability in practical samples by simulating the detection in real milk and drinking water samples. Importantly, our RAA-CRISPR/Cas12a detection system could complete the overall process (including extraction, amplification, and detection) within 55 min under optimized conditions, which is faster than most other reported sensors, which take several hours to several days. The signal readout could also be visualized by fluorescence generated with a handheld UV lamp or a naked-eye-detected lateral flow assay depending on the DNA reporters used. Because of the advantages of being fast, having high sensitivity, and not requiring sophisticated equipment, this method has a promising application prospect for in situ detection of trace amounts of pathogens.

Polyphenols and CRISPR as Quorum Quenching Agents in Antibiotic-Resistant Foodborne Human Pathogens (Salmonella Typhimurium, Campylobacter jejuni and Escherichia coli 0157:H7)

“…With the emerging technology of CRISPR Cas, the possibility of improving systems and devices for detecting microorganisms of interest in the food, medical and environmental industries becomes evident. Zhu L. et al, 2023, published the development of an ultrasensitive method for the detection of E. coli O157:H7 based on what they call RAA-CRISPR/12a (Recombinase-Aided Amplification, RAA), resulting in a highly efficient test that only requires 55 min to detect E. coli O157:H7 [89]. Recently, Jiang et al, 2023, developed a rapid, specific, and visual nucleic acid detection method called CRISPR/Cas12a-PMNT in Escherichia coli O157:H7, based on a combination of Cas12a techniques with RPA (Recombinase Polymerase Amplification) and cationic poly watersoluble [3-(3 ′ -N,N,N-triethylamino-1-propyloxy)-4-methyl-2,5-thiophene hydrochloride] (PMNT).…”

A multiplex RPA coupled with CRISPR-Cas12a system for rapid and cost-effective identification of carbapenem-resistant Acinetobacter baumannii

“…This property facilitates straightforward visual detection. Leveraging these features, the CRISPR-Cas12a system is particularly well-suited for bacterial detection and has rapidly advanced, demonstrating successful applications in detecting common pathogens such as Escherichia coli ( Zhu et al, 2023 ), Pseudomonas aeruginosa ( Liu et al, 2023 ), and Salmonella typhi ( Duan et al, 2024 ).…”