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

Cao X, Li S, Chen L, Ding H, Xu H, Huang Y, Li J, Liu N, Cao W, Zhu Y, Shen B, Shao N - Nucleic Acids Res. (2009)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Beijing Institute of Basic Medical Sciences, Beijing, China.

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.

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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.
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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.

Mentions: Because of a space-temporal antigenic expression model and high antigenic variation by bacteria, the detection of bacteria based on a single molecular probe may be prone to a false negative result. Therefore, combined detection of multiple molecular characteristics on bacteria with a panel of aptamers targeting different sites will improve the test sensitivity theoretically. The five aptamers were verified to recognize different molecular targets on S. aureus by a competition experiment (Supplementary Figure 2). The feasibility of using mixture of these five aptamers for recognizing S. aureus was then tested by flow cytometry and confocal microscopy in this study. Because we evaluate individual aptamer under concentration of 240 nM that is far higher than the concentration to induce maximum binding for anyone aptamer (Supplementary Figure 3), i.e. the corresponding target is saturated with each aptamer, we also use equimolarly mixed multiple aptamers with 240 nM each to gain a similar saturation state to evaluate the effect of mixture. The results indicated that mixed aptamer probes kept the specific property of an individual aptamer, i.e. distinguish S. aureus including 8325-4, 196 and 04018 from S. epidermidis, streptococcus and E. coli. (Figure 4A and B). The individual aptamer showed various degrees of recognition to different strains of S. aureus, which demonstrated a different expression profile by different strains (Figure 4A). Importantly, the combined application of aptamers showed enhanced fluorescence intensities, and thus a higher positive rate or binding capacity, against different strains of S. aureus in early growth state (Figure 5) and against 8325-4 of different growth states compared to the single probe (Figure 6), suggesting a more sensitive and effective strategy by multiple aptamers in bacteria identification. Because different aptamers recognize different targets and do not interfere with each other (Supplementary Figure 2); the EC50 value of each aptamer in the mixture will be the same as it is in the individual binding experiment. Therefore, the EC50 of the ssDNA in the mixture will be approximately the summation of EC50 of each aptamer theoretically. Accordingly, a mixture of aptamers with individual EC50s amongst 61.50–210.70 nM is characterized by an EC50 of 479.98 nM (Table 1).Figure 5.


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

Cao X, Li S, Chen L, Ding H, Xu H, Huang Y, Li J, Liu N, Cao W, Zhu Y, Shen B, Shao N - Nucleic Acids Res. (2009)

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.
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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.
Mentions: Because of a space-temporal antigenic expression model and high antigenic variation by bacteria, the detection of bacteria based on a single molecular probe may be prone to a false negative result. Therefore, combined detection of multiple molecular characteristics on bacteria with a panel of aptamers targeting different sites will improve the test sensitivity theoretically. The five aptamers were verified to recognize different molecular targets on S. aureus by a competition experiment (Supplementary Figure 2). The feasibility of using mixture of these five aptamers for recognizing S. aureus was then tested by flow cytometry and confocal microscopy in this study. Because we evaluate individual aptamer under concentration of 240 nM that is far higher than the concentration to induce maximum binding for anyone aptamer (Supplementary Figure 3), i.e. the corresponding target is saturated with each aptamer, we also use equimolarly mixed multiple aptamers with 240 nM each to gain a similar saturation state to evaluate the effect of mixture. The results indicated that mixed aptamer probes kept the specific property of an individual aptamer, i.e. distinguish S. aureus including 8325-4, 196 and 04018 from S. epidermidis, streptococcus and E. coli. (Figure 4A and B). The individual aptamer showed various degrees of recognition to different strains of S. aureus, which demonstrated a different expression profile by different strains (Figure 4A). Importantly, the combined application of aptamers showed enhanced fluorescence intensities, and thus a higher positive rate or binding capacity, against different strains of S. aureus in early growth state (Figure 5) and against 8325-4 of different growth states compared to the single probe (Figure 6), suggesting a more sensitive and effective strategy by multiple aptamers in bacteria identification. Because different aptamers recognize different targets and do not interfere with each other (Supplementary Figure 2); the EC50 value of each aptamer in the mixture will be the same as it is in the individual binding experiment. Therefore, the EC50 of the ssDNA in the mixture will be approximately the summation of EC50 of each aptamer theoretically. Accordingly, a mixture of aptamers with individual EC50s amongst 61.50–210.70 nM is characterized by an EC50 of 479.98 nM (Table 1).Figure 5.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Beijing Institute of Basic Medical Sciences, Beijing, China.

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.

Show MeSH
Related in: MedlinePlus