Staphylococcus haemolyticus is a Gram-positive coagulase-negative bacterium that is very common in hospitals. In recent years, hospital-acquired infections have received increasing attention due to their higher risk. Staphylococcus haemolyticus can further cause skin infections, meningitis, peritonitis, and even sepsis. Delays in clinical diagnosis can lead to worsening health status, so rapid diagnosis of S. haemolyticus is key to precise treatment.
The research team of Teacher Ji Tuo from the Central Laboratory Department of the Second People's Hospital of Lianyungang, China published an article "A rapid and visual detection of Staphylococcus haemolyticus in clinical specimens with MIRA-LFS" in the journal "Analytica Chimica Acta", with an impact factor of 6.2.
1.Research overview
In this study, a MIRA-LFS method based on the mvaA gene was established to detect Staphylococcus haemolyticus. The multi-enzyme isothermal rapid nucleic acid amplification technology MIRA was combined with lateral flow test strip LFS to achieve rapid pathogen detection.
The experiment was conducted at 37°C, and the results could be detected within 9 minutes. The detection sensitivity was 0.147CFU/reaction.
The MIRA-LFS detection method is suitable for the fixed-point detection of Staphylococcus hemolyticus in clinical samples, realizing the POCT method for identifying Staphylococcus hemolyticus, which is of great value for rapid disease diagnosis and treatment in areas with underserved medical services.
The research team used MIRA-LFS, qPCR and traditional bacterial culture methods to analyze 95 random clinical samples and confirmed the clinical applicability.
2.Research methods
The schematic diagram of MIRA-LFS detection is shown above.
The research used Amp-Future Biotech's DNA isothermal rapid amplification kit (colloidal gold test strip type) and nucleic acid detection test strips.
The mvaA gene primer length is 30-35 nucleotides, and the amplification product length is 100-350 bp. A total of 6 pairs of candidate primers were designed. MIRA analysis was performed using gDNA of standard Staphylococcus hemolyticus strains, candidate primers were screened, and amplification products were displayed using 2% agarose gel electrophoresis.
Then design the probe based on the forward primer sequence (extending 16 bp backward). The 5’ end of the probe was labeled with FITC and the 3’ end was labeled with C3 blocking. Use THF to replace the 31 nucleotide in the probe. THF is 30 bp from the 5’ end and 15 bp from the 3’ end. The 5’ end of the selected reverse primer is labeled with biotin.
Moreover, the research team optimized the reaction conditions for MIRA-LFS detection of Staphylococcus hemolyticus. Test at 37°C at intervals of 2 minutes from 4 to 12 minutes. The results are displayed at 8min reaction time and the test line is clearly visible. In addition, tests are conducted at intervals of 5°C from 22-52°C. 37-42℃ is the most suitable temperature for MIRA-LFS detection. Therefore, the optimal reaction time and temperature of the MIRA-LFS method for Staphylococcus haemolyticus are 8 min and 37°C, respectively. Together with the final result being visualized by LFS within 1 minute, the total detection time is 9 minutes.
3.Experimental results
Sensitivity verification
MIRA-LFS assay was performed on different concentrations of genomic DNA (gDNA) obtained from serially diluted Staphylococcus haemolyticus cultures (10^6-10^0 CFU/mL).
Probabilistic unit regression analysis showed that the 95% LOD determined by the MIRA-LFS assay was 0.147 CFU/reaction.
Specificity verification
By analyzing 18 common pathogenic microbial strains and 15 clinical Staphylococcus haemolyticus strains, the selectivity of MIRA-LFS detection of Staphylococcus haemolyticus was determined. The results showed that the MIRA-LFS test was negative for 18 other pathogenic bacteria and positive for 15 verified clinical samples of Staphylococcus haemolyticus.
MIRA-LFS detection is highly selective and suitable for screening of Staphylococcus hemolyticus.
Actual sample testing
Lianyungang Second People's Hospital randomly collected 95 specimens, including 35 skin and soft tissue samples, 16 urine samples, 14 sputum samples and 30 blood samples.
The results showed that the detection method was 100% consistent with the qPCR method. In addition, compared with the gold standard (GB/T4789.11-2003), the sensitivity and specificity of the MIRA-LFS detection method for identifying Staphylococcus hemolyticus infection were 100% and 98.73%, respectively. These results further verified the technical feasibility of this method in detecting Staphylococcus hemolyticus in clinical samples.
The MIRA-LFS method is used for rapid POCT detection of Staphylococcus hemolyticus. It is efficient, accurate, simple, and low-cost, and helps to make rapid diagnosis and treatment decisions.
Learn about MIRA technology
Amp-Future's multi-enzyme isothermal rapid nucleic acid amplification technology amplifies nucleic acids at a constant temperature of 39°C-42°C, with a reaction time of only 5-20 minutes. In addition, strict laboratory conditions are not required, and the application scenarios are diversified: general hospital laboratory departments, primary care, home testing, fields, etc.
Reagent product forms are diverse: freeze-dried powder, freeze-dried microspheres, etc.; display results are diverse: gel electrophoresis, fluorescence curve, colloidal gold test strips, etc. The reagent has stable performance, is easy to operate, and is convenient for storage and transportation.
With MIRA as the core platform technology, it has developed a series of supporting products such as "ultra-fast nucleic acid release technology", "dual-channel isothermal fluorescence detector", "integrated fully automatic closed detection system", "self-testing nucleic acid detection device", etc. Create an overall solution for rapid molecular detection.
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