Target nucleic acid amplification-free detection of Escherichia coli O157:H7 by CRISPR/Cas12a and hybridization chain reaction based on an evanescent wave fluorescence biosensor

Abstract

As the main foodborne pathogen, Escherichia coli O157:H7 poses a serious threat to public health. Herein, a target nucleic acid amplification-free method was developed integrating a hybridization chain reaction and CRISPR/Cas12a called the CHANCE (Cas12a-HCR evANescent wave fluorescenCE) system for the rapid and sensitive detection of E. coli O157:H7. The specific crRNA was designed with the rfbE gene of E. coli O157:H7 as the target. In the presence of the target, the trans-cleavage activity of Cas12a is activated and cleaves the HCR initiator, resulting in a decrease in the fluorescence signal. Under the optimized conditions, the CHANCE method could quantify E. coli O157:H7 with a concentration range from 10 to 108 CFU/mL, with a detection limit of 17.4 CFU/mL. By using the rapid extraction method, the accurate detection of E. coli O157:H7 can be achieved within 50 min and successfully applied in actual environmental water samples. Benefiting from free amplification and high flexibility, the CHANCE method provides an ideal alternative tool for the rapid and on-site detection of pathogenic bacteria in the environment and food.