A method for fluorescence detection of Staphylococcus aureus based on chimera sequence design and molecular beacons
A Staphylococcus, molecular beacon technology, applied in the direction of microbial-based methods, biochemical equipment and methods, microbial measurement/testing, etc., can solve the problems of incomplete extension, sequence replication pressure, genome instability, etc., and achieve the expansion of the detection range , Improve the detection sensitivity, the effect of strong specificity
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Embodiment 1
[0028] Example 1 The drawing of Staphylococcus aureus concentration standard curve
[0029] The sample containing Staphylococcus aureus was heated at 3000r·min -1 After centrifugation for 5 min, the pellet was washed 3 times with PBS buffer and resuspended in PBS buffer. 0.5μmol·L -1 The chimera sequence was denatured at 95°C for 5 minutes, then slowly cooled to 37°C and incubated for 45 minutes, mixed with 100 μL of PBS buffer containing different concentrations of Staphylococcus aureus, and placed in a shaker at 37°C, 220r min -1 Incubate for 45min. Take 10 μL of the above mixture, add 10 μL of molecular beacons and 5 μL of PBS buffer, and incubate in a 37° C. water bath for 100 min. Take 3 μL of the solution after incubation and add 30 μL restriction enzyme system, containing 6U Nb.bpu10I nicking endonuclease, 4.5U Bsm DNA polymerase and 250 μmol L -1 Free deoxyribonucleoside triphosphate, 10mmol L -1 Tris-HCl, 10mmol·L -1 MgCl 2 , 100mmol·L -1 KCl, 0.1 mg·mL -1...
Embodiment 2
[0031] The determination of Staphylococcus aureus content in the actual water sample of embodiment 2
[0032] In order to further verify the accuracy of this method in determining the content of Staphylococcus aureus in actual samples, Taihu Lake water and tap water without pretreatment were selected.
[0033] The cultured Staphylococcus aureus was heated at 3000r·min -1 Centrifuge for 5 min, and wash 3 times with PBS buffer to remove the medium. Then the precipitate was 8×10 2 CFU mL -1 and 8×10 4 CFU mL -1 The concentration was dissolved in 100 μL water sample. 0.5μmol·L -1 The chimera was denatured at 95°C for 5 minutes, then slowly cooled to 37°C and incubated for 45 minutes, mixed with 100 μL of PBS buffer containing different concentrations of Staphylococcus aureus, and placed in a shaker at 37°C, 220r min -1 Incubate for 45min. Take 10 μL of the above mixture, add 10 μL of molecular beacons and 5 μL of PBS buffer, and incubate in a 37° C. water bath for 100 mi...
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