Combining use of a panel of ssDNA aptamers in the detection of Staphylococcus aureus

Abstract

In this article, a panel of ssDNA aptamers specific to Staphylococcus aureus was obtained by a whole bacterium-based SELEX procedure and applied to probing S. aureus. After several rounds of selection with S. aureus as the target and Streptococcus and S. epidermidis as counter targets, the highly enriched oligonucleic acid pool was sequenced and then grouped under different families on the basis of the homology of the primary sequence and the similarity of the secondary structure. Eleven sequences from different families were selected for further characterization by confocal imaging and flow cytometry analysis. Results showed that five aptamers demonstrated high specificity and affinity to S. aureus individually. The five aptamers recognize different molecular targets by competitive experiment. Combining these five aptamers had a much better effect than the individual aptamer in the recognition of different S. aureus strains. In addition, the combined aptamers can probe single S. aureus in pyogenic fluids. Our work demonstrates that a set of aptamers specific to one bacterium can be used in combination for the identification of the bacterium instead of a single aptamer.

Figure 1.

Unequal strand length PCR using a primer with stem-loop structure. (A) The secondary structure of the (−) primer Pstemloop-3. (B) Schematic of PCR product. The (−) primer has a GC-rich reverse repeat sequence in the 5′ terminus, which can form the stem-loop structures and prevent the (+) strand elongation. (C) PCR products were analyzed by denaturing gel electrophoresis. Lane 1, the ssDNA of the naive library GP45; lane 2, the unequal length ssDNA products.

Figure 2.

The monitoring of enrichment of selection process by binding assay of radiolabeld pools with S. aureus. The number 0 represents the naive library and the numbers 1–7 represents the first to seventh round selected pool, respectively. Twenty-five picomoles of each radiolabeled pool were incubated with 10⁷ S. aureus 8325-4 for 45 min. The binding capacity was detected by scintillation counting.

Figure 3.

The binding capacity of candidate aptamers to S. aureus by flow cytometry analysis. The FITC-labeled individual aptamer was incubated with 10⁷ target bacteria (S. aureus 8325-4) or non-target bacteria (Streptococcus A5005) at 37°C for 45 min. The selected aptamers with higher ranked specificity and sensitivity were depicted with solid lines, and the GP45 naive library control and candidates with lower ranked specificity and sensitivity were depicted with dot lines. Representative results of three separate experiments.

Figure 4.

The characterization on specificity of individual aptamer and combined aptamers for S. aureus. The FITC-labeled individual aptamer or aptamer combination were incubated with 10⁷ bacteria at 37°C for 45 min. The concentration of aptamers in the binding buffer was 240 nM each. Representative results of three separate experiments. (A) Flow cytometry assay for the binding of aptamers with bacteria. The different color curves represent different strains of bacteria. (B) Overlaid confocal images of S. aureus 8325-4 and Sreptolococcus A5005 stained by aptamers from fluorescence and optical images. A 10 μm bar was shown.

Figure 5.

The comparison of sensitivity of combined aptamers to single aptamer for different S. aureus strains. Three different S. aureus strains in early log growth state were incubated with individual aptamer or aptamer combinations. The concentration of aptamer in the mixture was 240 nM each. Representative results of three separate experiments.

Figure 6.

The comparison of sensitivity of combined aptamers to single aptamer for S. aureus in different growth states. The S. aureus 8325-4 in different growth states were incubated with a single aptamer SA34 or aptamer combinations before flow cytometry analysis. The concentration of aptamer in the mixture was 240 nM each. Representative results of three separate experiments.

Figure 7.

The recognition of S. aureus in specimens of infectious diseases by aptamer mixture. The FITC labeled aptamer mixture was incubated with suppurative fluids smear on glass slides from patients of severe burn wound infection. The fluorescence patterns were observed with a confocal microscope. Upper row: S. aureus infection specimen; lower row: the specimen of mixed infection of Streptococcus and S. aureus.