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

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 similar to 1 CFU/mL (fluorescence method) and 1 x 10(2) 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 (10(3) CFU/mL) and ELISA (10(4)similar to 10(7) 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.

Automated summary

We developed a rapid and highly sensitive method for detecting E. coli O157:H7 using Recombinase-Aided Amplification (RAA) and CRISPR/Cas12a technology. This method showed high sensitivity, detecting as low as 1 CFU/mL (fluorescence method) and 1 x 10^2 CFU/mL (lateral flow assay) of E. coli O157:H7. The detection system could complete the entire process within 55 min and the signal readout could be visualized with a handheld UV lamp or a naked-eye-detected lateral flow assay. This method has a promising application prospect for in situ detection of trace amounts of pathogens due to its speed, sensitivity, and simplicity.