Efficient method for separating meticillin-resistant Sta-phylococcusaureus
A methicillin-resistant and staphylococcus-resistant technology, applied in the field of isolation of pathogenic bacteria based on nano-magnetic beads, can solve the problems of separation failure, high concentration of miscellaneous bacteria, poor monodispersity of micron magnetic beads, etc., to increase the chance of contact, Effects of improving separation efficiency and shortening separation time
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Embodiment 1
[0029] 1. The broom molecule-antibody complex is prepared according to the following steps:
[0030] (1) Weigh 1.0 mg of aminated broom molecules, suspend in 4 mL of phosphate buffer (PBS, 0.01 mol / L, pH 8.0), stir and add 545 μL of 25% glutaraldehyde aqueous solution dropwise to make glutaraldehyde The final concentration was 3%. React at room temperature for 3.5 h at a rotating speed of 150 r / min on a shaker;
[0031] (2) Add methicillin-resistant Staphylococcus aureus dropwise to the above solution MRSA Add 1 mL of specific antibody to make the final concentration reach about 3 mg / mL. React at room temperature for 24 hours at a shaking table of 150 r / min;
[0032] (3) The above solution was spin-dried under reduced pressure, dissolved in deionized water, and dialyzed in PBS and deionized water for 1 day; after the dialysis, the obtained solution was freeze-dried.
[0033] 2. The long-chain biotin-broom molecule-antibody complex is prepared according to the f...
Embodiment 2
[0039] Example 2 Enrichment effect experiment
[0040] (1) Take 1 mL of concentration as 10 4 cfu / mL MRSA Centrifuge at 12,000 rpm for 5 min in a 1.5 mL sterile centrifuge tube, discard the supernatant, and resuspend with an equal volume of sterile PBS solution.
[0041] (2) Enrichment and capture: respectively set the technical solution group of the present invention ( MRSA Antibody and long-chain biotin co-modified broom group), MRSA Specific antibody-modified nano-magnetic bead set, MRSA Specific antibody-modified micron magnetic bead group enriches target bacteria.
[0042] (3) After magnetic separation, pour the supernatant into a sterile centrifuge tube, and capture the MRSA The immunomagnetic beads were washed twice with PBST, mixed well, and the immunomagnetic bead complex was resuspended with 1 mL sterile PBS solution.
[0043] (4) Capture rate calculation: After gradient dilution of the enriched target bacteria resuspension in each group, count each gradi...
Embodiment 3
[0056] Example 3 Enrichment capture experiment
[0057] Conventional magnetic stand separation time is 30min, and all the other are with embodiment 2.
[0058] The catch rate of each group is as follows:
[0059] MRSA Capture efficiency of specific antibody-modified micron magnetic bead sets MRSA Capture efficiency of specific antibody-modified nanomagnetic bead sets MRSA Capture efficiency of broom groups co-modified with antibodies and long-chain biotin 56.7% 38.9% 91.2%
[0060] Experimental result shows, separates 3min among the comparative example 2, when separation time reaches 30min, the capture efficiency of three groups has all been improved, especially MRSA The capture efficiency of the specific antibody-modified nano-magnetic bead group is the most obvious, which shows that the capture efficiency of the nano-magnetic bead group can be greatly improved by extending the time, but it is still lower than the short-time separation (3mi...
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