A fluorescent aptasensor for emetic Bacillus cereus detection based on DNA-functionalized magnetic separation coupled with rolling circle amplification

Foodborne pathogens have been a serious threat to human health and safety for a long time. However, the complex food substrate undoubtedly increases the difficulty of bacterial detection at low concentrations. How to effectively improve the sensitivity of bacterial detection is a major problem that needs to be solved. In this study, the aptamer of emetic Bacillus cereus (B. cereus) was used as the recognition molecule to construct a magnetic capture probe. Utilizing the strong affinity between target bacteria and the aptamer, a fluorescence sensor based on aptamer-rolling circle amplification (Apt-RCA) was established for the detection of emetic B. cereus in milk spiked samples. In this study, DNA-functionalized magnetic beads were first synthesized. The strong affinity between the aptamer and the target bacteria makes it to be used not only as an antibody substitute for capturing the target bacteria, but also to compete the complementary DNA (cDNA) strand of the aptamer on the DNA-functionalized magnetic bead as a primer for the subsequent RCA reaction. RCA reaction can produce RCA products rich in a large number of G-quadruplex sequences. The fluorescent dye thioflavin T (ThT) can bind to the G-quadruplex structure and produce an obvious fluorescence signal. The fluorescence signal amplification strategy based on the magnetic separation platform and RCA reaction successfully realized the sensitive detection of emetic B. cereus in actual sample. Under optimal conditions, the limits of detection (LODs) of pure bacterial solution and milk samples were 1.4 × 102 CFU/mL and 2.2 × 103 CFU/mL, respectively. This method has the advantages of short detection time, simple operation and good specificity, which provides a new way of thinking for the detection of foodborne pathogens.