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

For rapid and sensitive detection of pathogenic bacterium P.eudomonas 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 (K_d), 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.