Establishment And Application Of Rapid Detection Of Staphylococcus Aureus Through Molecular Beacon-based Fluorescence In Situ Hybridization

Object:To establish a rapid molecular beacon-based fluorescence in situ hybridization method for direct identification of Staphylococcus aureus?SA?in positive blood culture or solid medium isolation samples.The fluorescent signal of SA was identified by fluorescence microscope or flow cytometry,and the clinical application of this method was evaluated.Methods:According to the 16S rRNA gene sequence of SA,three specific molecular beacon probes?MB1,MB2 and MB3?were synthesized,among which MB1 and MB2 were designed,and MB3 was the SA probe reported in literature.The signal-to-noise ratio?S/N?and thermal denaturation curves of three probes were evaluated,and the optimal probe was screened out.The optimization of the system includes molecular beacon concentration?0?mol/L²?mol/L?,MgCl₂ concentration?0mmol/L⁷mmol/L?and deionized formamide concentration?0%⁵5%?.A number of mismatched gene sequences with selected molecular beacons were designed,and the optimum hybridization temperature was screened by thermal denaturation curve and fluorescence derivative curve.Optimize the concentration of lysostaphin?0U/mL²0U/mL?to permeabilize the cell wall of SA.The system was used to detect 10 clinical isolates and standard strains?Gram-positive cocci?isolated from positive blood culture or solid medium,and the fluorescence signal was identified by fluorescence microscopy to evaluate the specificity of the system for SA detection.Using the SA standard strain,the negative blank SA bacteria liquid and the molecular beacon SA bacteria liquid were detected by flow cytometry,the difference of average fluorescence intensity between the two groups was analyzed.The negative blank SA bacterial solution,molecular beacon hybridization SA solution and non-specific fluorescent dye PI staining of SA solution were used as control.The positive cocci were detected by flow cytometry in 200 blood culture samples.Compared with the traditional phenotypic identification method,the specificity and sensitivity of this method for detecting SA were evaluated.Results:S/N of the designed and synthesized MB1 was significantly higher compared to MB2 and MB3?P<0.001?.S/N ratios of MB1 probe at different concentrations were all>20.The thermal denaturation curve of the probe showed that the fluorescence intensity of the molecular beacon remained stable at temperatures below 50??close to background fluorescence?,and the fluorescence intensity gradually increased from 50?to 65?,and the fluorescenc gradually decreased at greater than 65?.The established molecular beacon fluorescence in situ hybridization system was:1.4?mol/L MB1,2.5 mmol/L MgCl₂,25%deionized formamide.The thermal denaturation curve and fluorescence derivative curve show that when the temperature is below 45?,the MB1 can distinguish the two base mismatch gene sequences,and the single base mismatch gene sequence can be distinguished when the temperature is 45?⁵0?,and the single base mismatch gene sequence can be distinguished when the temperature is below 45?.The stability of MB1 is affected when the temperature is higher than 50?.The optimum permeabilization conditions for bacterial cell wall were:7.5 U/ml lysostaphin for 5 min;the optimized hybrid system was crossed with 10 isolates and standard strains isolated from positive blood culture or solid medium,Under the fluorescence microscope,only SA produced a fluorescent signal,and the rest showed no fluorescence.The average fluorescence intensity of molecular beacon hybridization SA solution?11.47±2.27?was higher than that of negative SA solution?1.71±0.59?by flow cytometry,P<0.001.The method was used to detect the positive cocci in 200 blood culture samples,the specificity and sensitivity were 100%and95.83%,respectively,which was extremely consistent with the traditional phenotypic identification method?Kappa value=0.967?.Conclusion:?1?The established molecular beacon-based fluorescence in situ hybridization method can rapidly identify SA of positive blood culture or solid medium growth by fluorescence microscopy or flow cytometry.?2?Based on the molecular beacon fluorescence in situ hybridization method,it can be applied to the detection of SA in clinical blood culture positive specimens with high specificity and sensitivity,and it is more consistent the traditional phenotypic identification method.