Selection, identification and application of DNA aptamers for the detection of Bifidobacterium breve

Abstract

In the present study, a single-stranded DNA (ssDNA) aptamer binding to Bifidobacterium breve with high avidity and selectivity was selected through a whole-bacterium-based systemic evolution of ligands using an exponential enrichment (SELEX) process. Following 12 rounds of selection specific for B. breve, three FAM-labeled aptamers were chosen for flow cytometry analysis and the results revealed that all three aptamers possessed a high binding affinity for B. breve. To obtain the optimal sequence, sequence truncation experiments of these three aptamers were conducted. An aptamer variant BB16-11f with high affinity and selectivity was acquired. In addition, the dissociation constant was significantly reduced to 18.66 ± 1.41 nM. Furthermore, an enzyme linked aptamer assay was developed to prove the potential application of the aptamer BB16-11f in the detection of B. breve. The results showed that the colorimetric assay had a linear relationship between the absorbance at 450 nm and the concentrations of B. breve ranging from 10³ cfu mL⁻¹ to 10⁷ cfu mL⁻¹ with a correlation coefficient of 0.98. The limit of detection (LOD) of the assay was 1000 cfu mL⁻¹. Additionally, the developed method was also successfully used to detect B. breve in a milk environment. Taken together, we hold that the developed colorimetric bioassay based on the aptamer BB16-11f is a promising method for the detection of B. breve.