Direct detection of Staphylococcus aureus in positive blood cultures through molecular beacon-based fluorescence in situ hybridization

Abstract

Objective: Clinical diagnosis of bloodstream infection diseases depends on the blood culture results. Bacterial identification by traditional methods is time-consuming. This study aimed to utilize molecular beacon-based fluorescence in situ hybridization (MB-FISH) for rapid and direct detection of Staphylococcus aureus in positive blood cultures.

Methods: Three molecular beacon probes (MB1, MB2 and MB3) were designed and synthesized to target the 16S rRNA gene fragment of S. aureus. The MB-FISH system was optimized, and the specificity of this method in detecting S. aureus was evaluated. This approach was used to test 41 g-positive clinical specimens with positive blood cultures. In addition, the consistency of this method with traditional methods was evaluated.

Results: Signal-to-noise ratio (S/N) of the molecular beacon MB1 was significantly higher than that of MB2 and MB3 (P < .001). The S/N ratios of MB1 probe at different concentrations were all >20. Thermal denaturation curve of the probe suggested that its hairpin structure can be opened and closed. Conditions such as deionized formamide concentration, ionic strength and temperature were optimized by monitoring the fluorescence intensity of MB1 in the presence or absence of its target sequence B1. The optimized hybridization system produced fluorescence only in S. aureus. The specificity and sensitivity of MB1 probe for detecting S. aureus in 41 specimens were 100% and 93.75%, respectively. Although sample size was small, MB-FISH appeared to be consistent with traditional culture methods (Kappa value = 0.948).

Conclusion: MB-FISH demonstrates strong specificity and high sensitivity, and can be used for direct detection of S. aureus in positive blood cultures.

Keywords: Blood culture; Bloodstream infection; Fluorescence in situ hybridization; Molecular beacons; Staphylococcus aureus.