Selection and Application of Specific Nucleic Acid Aptamers for the Detection of Pseudomonas aeruginosa

For rapid and sensitive detection of pathogenic bacterium Pseudomonas aeruginosa, the whole-cell systematic evolution of ligands by exponential enrichment (SELEX) technique was used to select single-stranded DNA (ssDNA) aptamers specific for P. aeruginosa. After 15 rounds of enrichment, 30 ssDNA sequences were obtained. By comparing the Gibbs free energy (ΔG) and dissociation constant (Kd), aptamer Apt13 was identified to have the best specificity and affinity for P. aeruginosa. P. aeruginosa labeled with fluorescein isothiocyanate (FITC) exhibited fluorescence, which could be quenched by the addition of Apt13. Counter-screening with other bacteria, including Escherichia coli, Staphylococcus aureus, Bacillus subtilis, and Micrococcus luteus, did not affect the fluorescence quenching effect of Apt13 on FITC-labelled P. aeruginosa. Based on this phenomenon, a fluorescence detection system for P. aeruginosa was developed. Whether or not P. aeruginosa is present in samples could be judged from the fluorescence quenching effect of Apt13. Under optimized concentration of Apt13 and incubation time of P. aeruginosa, the fluorescence quenching intensity exhibited a linear relationship with the concentration of P. aeruginosa ranging from 101 to 108 CFU/mL. The limit of detection (LOD) was 2 CFU/mL, and the entire detection process took less than 2.0 h. In conclusion, the developed method allowed effective detection of P. aeruginosa in water samples.