Flow Cytometric Detection of Bacillus spoOA Gene in Biofilm Using Quantum Dot Labeling

Quantum dot (QD)-induced fluorescence detection of bead-based DNA sandwich hybridization was studied for rapid analysis of the Bacillus spoOA gene in biofilms. Hybridization between two DNA probes and target DNA occurred and the hybridization signal was detected in a flow cytometer. To prepare the bead DNA capture and QD-DNA detection probes, the coupling or bioconjugation reactions were carefully controlled. It was successfully demonstrated that the fluorescence response of the hybrid complex was linear in the range of 3.2-1000 nM of synthetic target DNA (R-2 = 0.97) and that the detection limit was 0.02 nM. An optimized labeling method and bead-based DNA hybridization were then applied to real PCR products from a biofilm sample with satisfactory results (R-2 = 0.94), thereby confirming that the proposed assay provides a rapid, sensitive, and specific method for Bacillus spoOA gene detection in bofilms. This approach enables multiple target detection using multicolor QD-DNA probes in a shorter time.