Specific capture and detection of Staphylococcus aureus with high-affinity modified aptamers to cell surface...

Area of Science:

• Microbiology
• Biotechnology
• Molecular Biology

Background:

• Traditional methods for microbial contamination monitoring rely on culture, PCR, or antibodies.
• There is a need for novel reagents that offer high specificity and affinity for bacterial detection without cell lysis.

Purpose of the Study:

• To generate and characterize Slow off-rate modified aptamer (SOMAmer) reagents targeting Staphylococcus aureus cell surface proteins.
• To evaluate the utility of these SOMAmers for bacterial capture, enrichment, and direct detection.

Main Methods:

• SELEX (Systematic Evolution of Ligands by Exponential Enrichment) was employed to generate SOMAmers against Staphylococcus aureus surface proteins.
• Binding affinity and specificity were assessed using various assays.
• SOMAmers were utilized for bacterial cell capture, enrichment, and detection via qPCR and flow cytometry.

Main Results:

• High-affinity SOMAmers with sub-nanomolar dissociation constants (Kd) were generated for staphylococcal protein A (SpA) and clumping factors (ClfA, ClfB).
• Selected SOMAmers demonstrated specific binding to Staphylococcus aureus strains, with no cross-reactivity to other staphylococci or bacteria.
• SpA and ClfA SOMAmers effectively captured and enriched Staphylococcus aureus cells, improving detection limits and removing PCR inhibitors.
• Direct detection of Staphylococcus aureus was achieved using qPCR and fluorescence-labeled SOMAmers in flow cytometry.

Conclusions:

• SOMAmer reagents represent a promising novel class of tools for bacterial detection and capture.
• These aptamers enable sensitive and specific identification of Staphylococcus aureus without the need for culture or cell lysis.
• SOMAmers can be applied for capturing bacteria at low densities and for direct detection methods like qPCR and flow cytometry.