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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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Related in: MedlinePlus

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.
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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.

Mentions: The 3′(−) primer Pstemloop-3 consists of two compositions, the 5′-GC rich reverse repeat sequence with the ability to form stem-loop structures and the 3′-complement sequence of the template (Figure 1A). Due to the high Tm of the primer, the advanced structures still remain when the Taq DNA polymerase function at the temperature range of 55–65°C and thus prevent the elongation of the (+) strand (Figure 1B). Therefore, the PCR products exhibited two bands with different migration rates in denatured gel electrophoresis (Figure 1C); the lower band is at the same location level of the single strand template and the higher band is the extension product of Pstemloop-3 with reverse repeat sequence of 38 nt longer than the template.Figure 1.


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)

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.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2724295&req=5

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.
Mentions: The 3′(−) primer Pstemloop-3 consists of two compositions, the 5′-GC rich reverse repeat sequence with the ability to form stem-loop structures and the 3′-complement sequence of the template (Figure 1A). Due to the high Tm of the primer, the advanced structures still remain when the Taq DNA polymerase function at the temperature range of 55–65°C and thus prevent the elongation of the (+) strand (Figure 1B). Therefore, the PCR products exhibited two bands with different migration rates in denatured gel electrophoresis (Figure 1C); the lower band is at the same location level of the single strand template and the higher band is the extension product of Pstemloop-3 with reverse repeat sequence of 38 nt longer than the template.Figure 1.

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