A Method Based on a Modified Fluorescence In Situ Hybridization (FISH) Approach for the Sensing of Staphylococcus aureus from Nasal Samples.

Abstract

Staphylococcus aureus is a major source of bacteremia and develops several complications, causing high morbidity and mortality. Rapid identification and detection of these bacteria have become an important issue for biomedical applications. Herein, an optical method based on a modified fluorescence in situ hybridization (FISH) approach has been established using DNA hybridization technology for the swift detection of pathogenic S. aureus from clinical samples. The platform was constructed with single-stranded genomic DNA and microbial colony by directly immobilizing in agarose-polyvinyl alcohol (AG-PVA) hydrogel on the surface of a glass slide. The probe was based on an elongation factor encoding the tuf gene, which binds with equal affinity to single-stranded DNA targets as well as surface proteins on microbial cells. The probe was labeled with MFP488 fluorophore having excitation wavelength 501nm. The hybridization of the labeled probe with the target DNA and surface proteins was carried out under optimal FISH conditions, and the detection of bacteria was based on temporary field excitation of the labeled probe under a fluorescence microscope. Positive hybridization signals were detected by high fluorescence intensity. In comparison to genomic DNA, robust signals were observed with microbial cells, perhaps due to the moonlighting effect of the elongation factor Tu (Ef-Tu) expressed on the surface of bacterial cells. The applicability of the developed platform was tested on pediatric nasal samples, and results were verified with real-time qPCR. The designed platform is stable and sensitive, and after detailed optimization, a portable structure for on-site detection of pathogenic bacteria from clinical samples can be produced.