Staphylococcus haemolyticus is a gram-positive coagulase-negative bacterium that is very common in hospitals. In recent years, there has been increasing concern due to the high risk of hospital-acquired infections. Staphylococcus haemolyticus can further cause skin infections, meningitis, peritonitis, and even sepsis. Delays in clinical diagnosis can lead to deterioration in health conditions, so rapid diagnosis of Staphylococcus haemolyticus is key to precise treatment.
Professor Ji Tuo's research group from the Department of Central Laboratory of Lianyungang Second People's Hospital 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.
Overview of the study
In this study, Prof. Ji Tuo's group established a method for the detection of Staphylolyticus haemolyticus by MIRA-LFS based on mvaA gene, and used the multi-enzyme constant temperature rapid nucleic acid amplification technology MIRA combined with lateral flow test strip LFS to achieve rapid pathogen detection. The experiment was performed at 37°C and the results were detected within 9 min with a detection sensitivity of 0.147 CFU/reaction. The MIRA-LFS detection method is suitable for the fixed-point detection of Staphylococcus haemolyticus in clinical samples, and realizes the method of identification of Staphylococcus haemolyticus by POCT, which is of great value for the rapid diagnosis and treatment of diseases in underserved areas. The research group used MIRA-LFS, qPCR and traditional bacterial culture methods to analyze 95 random clinical samples, and confirmed the clinical applicability.
Research Methods
A schematic diagram of the MIRA-LFS test is shown above. In this study, AmpFuture Biotech's DNA constant temperature rapid amplification kit (colloidal gold test strip type) and nucleic acid detection test strip were used.
The primer length of mvaA gene was 30-35 nucleotides, and the length of the amplification product was 100-350 bp, and a total of 6 pairs of candidate primers were designed. The gDNA of standard Staphylolytic Staphylococcus strains was used for MIRA analysis, candidate primers were screened, and the amplified products were visualized by 2% agarose gel electrophoresis.
(Amplification products are displayed using agarose gel electrophoresis)
The probe is then designed according to the forward primer sequence (16 bp backward). The 5' end of the probe is labeled with FITC and the 3' end is labeled C3. The 31-position nucleotide in the probe is replaced with THF at a distance of 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.
(Schematic diagram of cross-dimer and sequence modification of primer-probe combinations)
In addition, the reaction conditions for the detection of MIRA-LFS by Staphylococcus haemolyticus were optimized. Tests are performed at 37 °C from 4-12 min at 2 min intervals. The results are shown in the 8 min reaction time, and the test line is clearly visible. In addition, the test is performed from 22-52 °C at 5 °C intervals. 37-42°C is the most suitable temperature for MIRA-LFS detection. Therefore, the optimal reaction time and temperature of the MIRA-LFS method for Staphylococcus haemolyticus were 8 min and 37 °C, respectively. Together with the fact that the final result is visualized by LFS within 1 min, the total detection time is 9 min.
(Optimization of reaction time and temperature)
Experimental results
1. Sensitivity verification
Different concentrations of genomic DNA (gDNA) obtained from serial dilutions of Staphylococcus haemolytica cultures (10^6-10^0 CFU/mL) were tested for MIRA-LFS.
(Positive signal at different concentrations)
PROBABILISTIC UNIT REGRESSION ANALYSIS SHOWED THAT THE 95% LOD DETERMINED BY MIRA-LFS ASSAY WAS 0.147 CFU/REACTION.
(Limit of Detection Calculation)
2. Specificity validation
The selectivity of MIRA-LFS detection was determined by analyzing 18 strains of common pathogenic microorganisms and 15 strains of clinical Staphylococcus haemolyticus. The results showed that the MIRA-LFS test was negative for all 18 other pathogenic bacteria, and positive for 15 verified clinical samples of Staphylococcus haemolyticus.
(8 strains of standard pathogenic bacteria and 15 strains of clinical hemolytic streptococcus were tested, respectively)
The MIRA-LFS assay has high selectivity and is suitable for the screening of Staphylococcus haemolyticus.
3. Actual sample testing
A total of 95 samples were randomly collected from Lianyungang Second People's Hospital, including 35 samples of skin and soft tissue, 16 samples of urine, 14 samples of sputum and 30 samples of blood.
(95 clinical specimens were tested by different methods)
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 assay for the identification of Staphylococcus haemolytica infection were 100% and 98.73%, respectively. These results further validate the technical feasibility of the method in the detection of Staphylococcus haemolyticus in clinical samples.
The MIRA-LFS method is used for the rapid detection of Staphylococcus haemolyticus by POCT, which is efficient, accurate, simple, and cost-effective, which is helpful for rapid diagnosis and treatment decisions.
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